JPH11508445A - Cytokines that induce apoptosis - Google Patents

Abstract

  (57) [Summary] A novel cytokine called TRAIL induces apoptosis of certain target cells, including cancer cells and virus infected cells. An isolated DNA sequence encoding TRAIL is disclosed, along with expression vectors and transformed host cells useful in producing TRAIL polypeptide. Antibodies that specifically bind TRAIL are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION                    Cytokines that induce apoptosis                                Background of the Invention   Programmed cell death, also known as apoptosis, is a combination of necrosis and cell death. Is different. Apoptosis is associated with embryogenesis, metamorphosis, endocrine-dependent tissue atrophy, Of immune thymocytes (by their antigen-receptor complex also Occur in death (induced by glucocorticoids) (Itoh et al., Cell 66: 233). , 1991). During maturation of T cells in the thymus, T cells that recognize self-antigens It is destroyed by the torsion process, but others are just selected. Some kind of self-epitopes Some T cells (eg, disabled and given a given self- (Provided with protein determinants) escaped this elimination process and continued It has been suggested that it may play a role in autoimmune diseases (Gammon et al., Imm. unology Today 12: 193, 1991).   A cell surface antigen known as Fas has been reported to mediate apoptosis And plays a role in clonal elimination of autoreactive T cells (Itoh et al., Cell 66: 233, 1991; Watanabe-Fukunaga et al., Nature 356: 314, 1992). Cross-linking of a specific monoclonal antibody to Fas can affect a variety of cell lines. It is reported to cause potosis (Yonehara et al., J. Exp. Med., 169: 1747 Trauth et al., Science, 245: 301, 1989). However, under certain conditions The binding of the specific monoclonal antibody to Fas was May have a costimulatory effect on T cells (Alderson et al., J. Exp. Med., 178: 2231, 1993).   Rat Fas ligand (Suda et al., Cell, 75: 1169, 1993) and human Fas Riga (Takahashi et al., International Immunology 6: 1567, 1994). DNA has been isolated. Fas ligand for cells expressing Fas antigen Binding has been demonstrated to induce apoptosis (Suda et al., Supra, and T akahashi et al., supra).   The presence or absence of one or more other molecules that play a role in apoptosis And identification studies are desired. Identification of such molecules regulates apoptosis It will provide yet another means, and furthermore the development and self-tolerance of the immune system. And provide further insights into the etiology of autoimmune diseases.                                Summary of the Invention   The present invention relates to a novel cytokine protein, And an expression vector comprising the isolated DNA. Tumor Sex of a novel cytokine that is a member of the tumor necrosis factor (TNF) family of ligands Quality includes the ability to induce apoptosis of certain types of target cells. But Thus, this protein is used as a TNF-related apoptosis-inducing ligand (TRAIL) Called. Some types of cells that die upon contact with TRAIL include leukemia, There are cancer cells, such as lymphoma and melanoma cells, and cells infected by the virus.   A method for producing a TRAIL polypeptide may be performed under conditions suitable for TRAIL expression. Using a recombinant expression vector containing DNA encoding TRAIL After transforming the cells, the expressed TRAIL polypeptide is recovered from the culture. Do that. Also provided are antibodies directed against the TRAIL polypeptide .                             BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the results of the assay described in Example 8. The assay uses soluble human TRA IL polypeptide induced death of Jurkat cell line, a leukemia cell line showed that.   FIG. 2 shows the results of the assay described in Example 11. Soluble human TRAIL polypeptide Contact with peptides induces death of human fibroblasts infected with cytomegalovirus Did not kill non-viral infected fibroblasts.                             Detailed description of the invention   Here, a novel protein, called TRAIL, encodes TRAIL Along with the recombinant expression vector containing the DNA and TRAIL DNA. Offer. Methods for producing recombinant TRAIL polypeptides are useful for expressing TRAIL. Culturing a host cell transformed with the recombinant expression vector under appropriate conditions; And recovering the expressed TRAIL.   The present invention also provides an antibody that specifically binds a TRAIL protein. one In one embodiment, the antibodies are monoclonal antibodies.   TRAIL proteins are not limited to cancer cells and viral Induces apoptosis in certain types of target cells, such as transformed cells, including stained cells . As demonstrated in Examples 5, 8, 9 and 10 below, TRAIL Induces apoptosis of leukemia, lymphoma and melanoma cell lines. TRAIL's Some uses are in killing cancer cells. TRAIL further Used in the treatment of ills infection. Cytomegalovirus (CMV) infection Make fibroblasts susceptible to apoptosis when contacted with TRAIL. However, uninfected fibroblasts did not die upon contact with TRAIL (actual See Example 11).   Isolation of DNA encoding human TRAIL is described in Example 1 below. . The nucleotide sequence of human TRAIL DNA isolated in Example 1 is shown in SEQ ID NO: : 1 and the amino acid sequence encoded thereby is shown in SEQ ID NO: 2. You. This human TRAIL protein has an N-terminal cytoplasmic domain (amino acids 1-1). 8), the transmembrane part (amino acids 19-38) and the extracellular domain (amino acids 39 To 281). The extracellular domain has a receptor binding moiety.   Large intestine transformed with a recombinant vector containing this human TRAIL DNA The E. coli strain DH10B cells were obtained on June 14, 1995 by American type. ・ Deposited with American Type Culture Collection , And accession number 69849. The deposit is made under the terms of the Budapest Treaty. It was done according to. The recombinant vector in the deposited strain was described in (Example 5). Ii) Expression vector pDC409. The vectors are SalI and NotI. And a human TRAI containing the complete coding region set forth in SEQ ID NO: 1. LDNA was ligated into the vector.   The DNA encoding the second human TRAIL protein is described in Example 2. Isolated. The nucleotide sequence of this DNA is shown in SEQ ID NO: 3, and The amino acid sequence encoded thereby is shown in SEQ ID NO: 4. Its coded The protein consists of an N-terminal cytoplasmic domain (amino acids 1-18), a transmembrane Nos. 19-38) and the extracellular domain (amino acids 39-101).   Since the DNA of SEQ ID NO: 3 lacks a part of the DNA of SEQ ID NO: 1, It is called the human TRAIL deletion mutant (huTRAILdv) clone. Array number Nucleotides 18 to 358 of No. 1 correspond to huTRAILdvDN of SEQ ID NO: 3. Identical to nucleotides 8-348 of A. Nucleotide 359 of SEQ ID NO: 1 ~ 506 is missing from the cloned DNA of SEQ ID NO: 3. The deletion is Causes transposition in the reading frame, resulting in amino acid 101 of SEQ ID NO: 4. Followed by an in-frame stop codon. Therefore, the DNA of SEQ ID NO: 3 is Encodes a partially truncated protein. Amino acids 1 to 9 of SEQ ID NO: 2 0 is identical to amino acids 1 to 90 of SEQ ID NO: 4. However, its deletion For the C-terminal part of the huTRAILdv protein (amino acid of SEQ ID NO: 4) Acids 91-101) are different from the residue at the corresponding position in SEQ ID NO: 2. Full length In contrast to the huTRAIL protein, the truncated huTRAILdv Protein induces apoptosis in T-cell leukemia cells of the Jurkat cell line Does not show the ability to   DNA encoding mouse TRAIL protein is also described in Example 3. Was isolated as follows. The nucleotide sequence of this DNA is shown in SEQ ID NO: 5, and The amino acid sequence encoded thereby is shown in SEQ ID NO: 6. That code The isolated protein has an N-terminal cytoplasmic domain (amino acids 1-17), a transmembrane (Amino acids 18-38) and extracellular domain (amino acids 39-291) . This mouse TRAIL is a human TRA of SEQ ID NO: 2 at the amino acid level. 64% identical to IL. The coding region of the mouse TRAIL nucleotide sequence is 75% identical to the coding region of the human nucleotide sequence of SEQ ID NO: 1.   One embodiment of the present invention provides an N-terminal amino acid sequence MetAlaMetMetGluValGlnGlyG. lyProSerLeuGlyGlnThr (amino acids 1 to 15 of SEQ ID NOs: 2 and 4) Human TRAIL protein. N-terminal amino acid sequence MetProSerSerGlyAl aLeuLysAspLeuSerPheSerGlnHis (amino acids 1 to 15 of SEQ ID NO: 6) A mouse TRAIL protein is also provided herein.   The TRAIL protein of the present invention is a protein known as Fas ligand. (Suda et al., Cell, 75: 1169, 1993; Takahashi et al., International Immu nology 6: 1567, 1994). Fas ligand is activated by a receptor known as Fas. Induces apoptosis in some cell types. As demonstrated in Example 5, TRAIL-induced apoptosis of target cells is not mediated by Fas. Array The human TRAIL amino acid sequence of No. 2 is shown in Takahashi et al., Supra. Approximately 20% identical to the human Fas ligand amino acid sequence. Human TRAIL cells The ectodomain is approximately 28.4% identical to the extracellular domain of human Fas ligand .   The amino acid sequences disclosed herein show that TRAIL is a ligand of the TNF series (Smith et al., Cell, 73: 1349, 1993; Suda et al., Cell, 7). 5: 1169, 1993; Smith et al., Cell, 76: 959, 1994). Human TRAIL extracellular domain Between the amino acid sequence and the amino acid sequences of the extracellular domains of other proteins in this series The percent identity between is as follows: 28.4% Fasligant, lymphotoxin 22.4% for -β, 22.9% for TNF-α, 23.1% for TNF-β, CD3 22.1% for 0 ligand and 23.4% for CD40 ligand.   Examining TRAIL for its ability to bind to receptors of the TNF-R series of receptors Was. Binding analysis was performed using the slide autoradiograph of Gearing et al. (EMBO J. 8: 3667; 1989). This was done using the luffy procedure. The analysis showed that human CD30, CD40, 4-1 BB, OX40, TNF-R (p80 type), CD27 or LTβR (TNFR -Also known as detectable binding of human TRAIL to RP) Was. The results of Example 5 show that human TRAIL does not bind human Fas ing.   TRAIL polypeptides of the present invention include those set forth in SEQ ID NOs: 2 and 6. And polypeptides having amino acid sequences that are very homologous. By way of example and not limitation, homologues, mutations from other mammalian species Body (both naturally occurring variants and those produced by recombinant DNA technology), And TRAIL fragments that retain the desired biological activity . Such polypeptides include the TRAIL proteins of SEQ ID NOs: 2 and 6. Exhibits biological activity and is preferably represented by SEQ ID NO: 2 or SEQ ID NO: 6 At least 80% identical to the amino acid sequence (most preferably at least 90% identical) ) Including the amino acid sequence These embodiments of the invention are described in further detail below. You.   Conserved sequences located in the C-terminal portion of proteins of the TNF series are incorporated herein by reference. Smith et al. (See Cell, 73: 1349, 1993, p. 1353 and FIG. 6). Suda et al. (Cell, 75: 1169, 1993; see FIG. 7); Smith et al. (Cell, 76:95 9, 1994, see FIG. 3); and Goodwin et al. (Eur. J. Immunol. 23: 2631, 1). 993, see FIG. 7 and pages 2638-39). Saved ( Amino acids of human TRAIL protein (at least for most members of the TNF series) Among the acids, positions 124 to 125 (AH), 136 (L), and 15 of SEQ ID NO: 2 4th (W), 169th (L), 174th (L), 180th (G), 182th (Y ), 187 (Q), 190 (F), 193 (Q) and 275-276 (FG). Another structural feature of TRAIL is that the transmembrane C Between the terminus and the portion of the extracellular domain considered to be most important for biological activity It is a spacer part between them. This spacer located at the N-terminus of the extracellular domain The portion consists of amino acids 39-94 of SEQ ID NO: 2. Similar spacers Family members, such as the CD40 ligand. Amino acids 138-1 53 is a loop between the β-sheets of the folded (three-dimensional) human TRAIL protein. Equivalent to   Provided herein are membrane-bound TRAIL proteins (cytoplasmic domain , Transmembrane and extracellular domains), and full-length TRAIL proteins TRAIL fragments that retain the desired biological properties of the protein. one In one embodiment, the TRAIL fragment contains all or an extracellular domain. Contains a portion, but is thought to retain the polypeptide on the cell membrane A soluble TRAIL polypeptide lacking a porcine moiety. Soluble TRAIL Proteins can be secreted from the cells in which they are expressed. Conveniently The heterologous signal peptide is N-terminal such that soluble TRAIL is secreted during expression. Fused to the end.   Soluble TRAIL can be used to identify intact cells expressing the desired protein, for example. Can be separated from the culture medium by centrifugation and the presence of the desired protein It can be confirmed by assaying the medium (supernatant) (and its insoluble membrane formation). Can be distinguished from the combined counterpart). The presence of TRAIL in the medium indicates that the protein From the TRAIL protein It is. Naturally occurring soluble forms of TRAIL are encompassed by the present invention.   The use of soluble forms of TRAIL is advantageous for some applications. Recombinant host cells Purification of proteins from vesicles is easy as soluble proteins are secreted from the cells become. Furthermore, soluble proteins are generally more suitable for intravenous administration.   Examples of soluble TRAIL polypeptides include the complete extracellular domain (eg, sequence (Amino acids 39 to 281 of SEQ ID NO: 2 or amino acids 39 to 291 of SEQ ID NO: 6) It contains. Of extracellular domains that retain the desired biological activity Fragments are also provided. Such fragments advantageously include the above Contains a portion of TRAIL that is conserved in the TNF family of ligand proteins. I will.   Yet another example of a soluble TRAIL polypeptide is the cytoplasmic domain and transmembrane Not all or part of the above spacer part as well as the part It is. Thus, soluble human TRAIL polypeptides are But not including polypeptides comprising amino acids x-281, wherein x represents any one of amino acids 39 to 95 of SEQ ID NO: 2. Residue 95 is N In embodiments that are terminal amino acids, the complete spacer portion has been deleted.   TRAIL fragments, including soluble polypeptides, can be obtained using a number of conventional techniques. Can be produced by any of the above methods. The desired TRAIL fragment The coding DNA sequence is used to express the expression vector for the production of the TRAIL fragment. May be subcloned into the sample. The DNA sequence encoding TRAIL is And, advantageously, the sequence encoding the appropriate leader or signal peptide. Are combined. A DNA fragment encoding the desired TRAIL can be obtained by known techniques. It can be synthesized economically using the method. DNA fragments are also full-length Produced by restriction endonuclease digestion of the cloned NDA sequence and It can be isolated by electrophoresis on a galose gel. 5 'or 3' if necessary Oligonucleotides that reconstitute the ends to the desired point are digested with restriction enzymes. Living The same DNA fragment may be ligated. Oligonucleotides like this Further comprises a restriction endonuclease cleavage site upstream of the desired coding sequence, An initiation codon (ATG) can then be placed at the N-terminus of the coding sequence.   Well known polymerase chain reaction (PCR) procedures are also used to Used to isolate and amplify the DNA sequence encoding the fragment. it can. Oligonucleotides that determine the desired end of the DNA sequence are 5 'and And 3 'primer. The oligonucleotide is further restricted Expression of an amplified DNA fragment containing an endonuclease recognition site Insertion into a vector can be facilitated. PCR technology is available from Saiki et al., Scie. 239: 487 (1988); Recombinant DNA Methodology, supervised by Wu et al., Academic Press , Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to  Methods and Applications, supervised by Innis et al., Described in Academic Press, Inc. (1990) Have been.   As will be appreciated by those skilled in the art, each of the TRAIL proteins and Each transmembrane segment is a conventional format for identifying its type of hydrophobic domain. Identified by standard. The exact boundaries of the transmembrane part are different from those shown above. Can differ slightly (probably up to 5 amino acids at each end) . Computer programs available to identify such hydrophobic moieties are available It is possible.   The TRAIL DNA of the present invention includes cDNA, chemically synthesized DNA, and PCR. DNA, genomic DNA and combinations thereof. Get Nom TRAIL DNA is prepared using the TR disclosed herein using standard techniques. Can be isolated by hybridization to AIL cDNA You. RNA transcribed from TRAIL DNA is also encompassed by the present invention. You.   A search of the NCBI databank found that the TRAIL DNA had the same portion as 5 Different expressed sequence tags (ESTs) were identified. These ESTs (deposited by NCBI) Nos. T90422, T82085, T10524, R31020 and Z36726) are all human cDN A fragment. The NCBI record contains any EST coded Does not reveal the polypeptide, and if so, what its reading frame could be Is not shown. However, with the disclosure of the complete TRAIL coding region, Even if the EST is expressed using the information in the reading frame indicated in the book, A polypeptide of the TRAIL polypeptide described in the claims. No one will have toxogenic properties. In other words, five types of ESTs When inserted into an expression vector downstream from the initiation methionine codon, In the reading frame described in the text, the resulting expressed polypeptide contains Sufficient of the extracellular domain of TRAIL to induce Nothing would include the part.   Some embodiments of the present invention relate to nucleotides 88-933 of SEQ ID NO: 1 ( Human TRAIL coding region); nucleotides 202 to 933 of SEQ ID NO: 1 (human TRAIL encoding the extracellular domain); nucleotides of SEQ ID NO: 5 47-922 (mouse TRAIL coding region); and the nucleoside of SEQ ID NO: 5 From Tides 261 to 922 (encoding the mouse TRAIL extracellular domain) An isolated DNA comprising a nucleotide sequence selected from the group consisting of: Arrangement Encoding biologically active fragments of the proteins of SEQ ID NOs: 2 and 6 Provided DNA is also provided. In yet another embodiment, the nucleotide sequence of SEQ ID NO: 1 No. 370-930 and a sequence comprising nucleotides 341-919 of SEQ ID NO: 5 Which are the specific human and murine soluble TRAIs described in Example 7. Each encodes an L polypeptide.   Due to the degeneracy of the genetic code, the two DNA sequences can differ, Encodes the same amino acid sequence. The present invention therefore relates to natural human or DNA or fragment thereof containing the coding region of P. TRAIL cDNA And degenerate as a result of the genetic code to the native TRAIL DNA sequence Isolated, encoding a biologically active TRAIL selected from DNA DNA sequences are provided.   Further provided herein are purified TR, both recombinant and non-recombinant. AIL polypeptide. Natural TRA retaining desired biological activity Mutants and derivatives of the IL proteins are also within the scope of the present invention. One fruit In embodiments, the biological activity of the TRAIL variant is a native TRAIL protein. It is essentially equivalent to the biological activity of proteins. One desired biology of TRAIL An activity is the ability to induce the death of Jurkat cells. Apoptosis of target cells Assays for detecting viruses are well known. DNA laddering is an appointment Included in the characteristics of cell death by torsion and reduce apoptotic cell death to necrotic It is recognized as one of the observable phenomena that distinguishes from bleeding. The death of the target cell Examples of suitable assay techniques for detecting apoptosis include Examples 5 and 8- 11 are described. Another property of TRAIL is that The ability to bind to cells.   TRAIL variants include, for example, mutations of the native TRAIL nucleotide sequence. Can be obtained by The TRAIL variants discussed herein are naturally-occurring A polypeptide substantially homologous to TRAIL of one or more of Amino acids different from those of native TRAIL due to deletion, insertion or substitution of Has an array. The DNA sequence encoding the TRAIL of the present invention may be a native TR The addition of one or more nucleotides when compared to the AIL DNA sequence. Sequences that include additions, deletions or substitutions, but are essentially identical to the native TRAIL protein. Encompasses said sequence encoding a TRAIL protein that is biologically equivalent to You.   The variant amino acid or DNA sequence preferably has a minor sequence with the native TRAIL sequence. At least 80% identical, most preferably at least 90% identical. Natural and And the degree of homology (% identity) between the mutant and mutant sequences, for example, is generally determined for this purpose. By comparing the two sequences using the computer program used, Can be determined. One suitable program is Devereux et al. (Nucl.Acid Res. 12: 387,1984) and described at the University of Wisconsin Genetics Compu. GAP computer program, available from the UWGCG Version 6.0. The GAP program includes Smith and Waterman (Adv .Appl.Math 2: 482,1981), as modified by Needleman and Wunsch (J. Mol. Biol. 48: 443, 1970). In short, GAP program Is the number of matching parallel symbols (ie, nucleotides or amino acids) divided by two Distribution Determine identity as the total number of shorter symbols in a column. GAP program preferred The implicit parameters include (1) unary comparison matrices for nucleotides. Rix (including 1 for identical and 0 for non-identical) and Schwartz and D supervised by ayhoff, Atlas of Protein Sequence and Structure, National Biomedical  Gribskov et al., As described by the Research Foundation, pages 353-358, 1979. And Burgess, Nucl. Acids Res. 14: 6745,1986, weighted comparison matrix; (2) 3.0 penalty for each gap and the respective An additional 0.10 penalty for the symbol; and (3) a terminal gap penalty. No tee, included.   Alterations of the natural amino acid sequence can be accomplished by any of a number of known techniques. You. Mutation flanked by restriction sites that can be ligated to fragments of the native sequence Specific inheritance by synthesizing modified mutant sequence-containing oligonucleotides Can be introduced to the child. After ligation, the resulting rearranged sequence contains the desired amino acid Encodes analogs with insertions, substitutions or deletions.   Alternatively, oligonucleotide-directed site-directed mutagenesis The specific codons altered by the necessary substitutions, deletions or insertions. An altered gene having the same can be provided. Technology to make such changes (Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (B ioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principle) and Methods, Plenum Press, 1981); and U.S. Patent Nos. 4,518,584 and No. 4,737,462, which are incorporated herein by reference.   Variants can include conservatively substituted sequences, and can include native TRAIL poly One or more amino acid residues of the peptide are replaced by different residues However, the conservatively substituted TRAIL polypeptide is a native TRAIL polypeptide. Has been shown to retain the desired biological activity essentially equivalent to that of tide. You. Examples of conservative substitutions include altering the secondary and / or tertiary structure of TRAIL. Some amino acid substitutions are not allowed. In another example, the desired biological activity is on a target cell. Ability to bind to a receptor and induce apoptosis of its target cells In some cases, amino acid substitutions outside the receptor binding domain are included. Given Amino acids can be replaced with residues having similar physical and scientific properties. For example, replacing one aliphatic residue with another (such as Ile, Val, Leu or Ala) Can be substituted for each other) or one polar residue can be replaced by another (Lys and Arg Glu and Asp; or between Gln and Asn). Other like this Such conservative substitutions, for example, substitution of an entire moiety having similar hydrophobicity, are well known. Save TRAIL polypeptides containing amino acid substitutions are required to produce the desired native TRAIL. Assays described herein to confirm retention of physical activity Can be tested in one of the TRAI containing such conservative amino acid substitutions DNA encoding an L polypeptide is encompassed by the present invention.   Located at the C-terminal part of TNF series proteins and important for biological activity Conserved amino acids that are believed to be have been identified. These conserved sequences are described in Smith et al. (Ce ll, 73: 1349, 1993, p. 1353 and FIG. 6); Suda et al. (Cell, 75: 1169). 1993, FIG. 7); Smith et al. (Cell, 76: 959, 1994; see FIG. 3). And Goodwin et al. (Eur. J. Immunol. 23: 2631,1993, FIG. 7 and 2638-39). Page). Conveniently, the conserved amino acids have a conservative substitution sequence. If it does, it does not change. If changed, the equiposition of other members of the TNF series The amino acids found in are replaced.   TRAIL also contains other components such as glycosyl groups, lipids, phosphates, acetyl groups, etc. Induces TRAIL by forming a covalent or agglutination bond with a chemical residue It can be modified to produce a body. The covalent derivative of TRAIL is T To functional groups on RAIL amino acid side chains or to TRAIL polypeptide or By attaching a chemical residue to the N-terminus or C-terminus of its extracellular domain. Can be manufactured. Other derivatives of TRAIL within the scope of the present invention include N-terminal Other proteins, such as by synthesis in recombinant culture, such as terminal or C-terminal fusions Or a covalent or aggregated conjugate of TRAIL with a polypeptide or Fragments are included. For example, the conjugate is the N-terminus of the TRAIL polypeptide A signal or leader polypeptide sequence (eg, Saccharomyces (Sacc) haromyces). The signal or Lee Dipeptides from their synthesis site to sites inside or outside the cell membrane or cell wall of Governance of the conjugate is governed by co-translation or post-translation.   TRAIL polypeptide fusions facilitate TRAIL purification and identification. And may be added to the peptide. Such peptides include, for example, No. 5,011,912 and Hopp et al., Bio / Technology 6: 1204, 1988. One such pepti Do No. 7), which is a highly antigenic and specific monoclonal antibody To give an epitope that is reversibly bound by the recombinant protein expressed. Allows for rapid assay of quality and easy purification. This sequence also contains the bovine mucosal It is specifically cleaved by telokinase at the residue immediately after Asp-Lys pairing. This page Peptide-capped fusion proteins are also suitable for intracellular degradation in E. coli. Can be resistant.   A murine hybridoma designated 4E11 is described in (US Pat. No. 5,011,912). In the presence of certain divalent metal cations, the peptide DYKDDDDK (SEQ ID NO: No. 7), and produce a monoclonal antibody Ruchi Expression systems useful for producing recombinant proteins fused to Monochromes useful for binding peptides and purifying the recombinant protein Null antibodies are available from Eastman Kodak Company, Scientific Imaging Systems, New York. Available from Bun, Connecticut.   The present invention further provides for T with or without associated native pattern glycosylation. RAIL polypeptide. TRAI expressed in yeast or mammalian expression systems L depends on the choice of the expression system, the TR of native molecular weight and glycosylation pattern It can be similar or significantly different from the AIL polypeptide. colon Expression of a TRAIL polypeptide in a bacterial expression system, such as a fungus, may be a non-glycosylated molecule. give.   Glycosylation sites in the TRAIL extracellular domain may be yeast or mammalian expression A system for expressing uniform reduced carbohydrate analogs using the system It can be modified to prevent lycosylation. N-glycosyl in eukaryotic polypeptides The site of oxidation is characterized by the amino acid triplet Asn-XY, where X Is any amino acid except Pro and Y is Ser or Thr. Suitable modifications to the nucleotide sequence encoding this triplet are cause substitutions, additions or deletions that prevent the attachment of carbohydrate residues at the sn side chain. There will be. Known methods for inactivating N-glycosylation sites in proteins include rice. No. 5,071,972 and those described in EP 276,846. Potential N-glycosylation sites are from 109 to 109 in the human protein of SEQ ID NO: 2. Found at positions 111 and 52-54 in the murine protein of SEQ ID NO: 6 .   In another example, encoding a Cys residue that is not essential for biological activity Existing sequence may have its Cys residue deleted or replaced with another amino acid. To prevent incorrect intramolecular disulfide bridge formation upon reconstitution Can be. Other variants are expressed in yeast systems where KEX2 protease activity is present. Produced by modification of adjacent dibasic amino acid residues to promote European Special No. 212,914 discloses a KEX2 protease processing site in a protein. Disclosed is the use of site-directed mutagenesis to inactivate. KEX2 Protea Zeprocessing sites alter the Arg-Arg, Arg-Lys and Lys-Arg pairs. Inactivation by deleting, adding or substituting residues as in Eliminates the generation of basic residues. Lys-Lys pairing is almost insensitive to KEX2 cleavage Not susceptible, and the conversion of Arg-Lys or Lys-Arg to Lys-Lys is Figure 2 shows a conservative and preferred approach to inactivation of the X2 site. Potential KEX2 Pro The protease processing site is located at positions 89-90 in the protein of SEQ ID NO: 2. And positions 149 to 150 and positions 85 to 86, 13 in the protein of SEQ ID NO: 6. Found at positions 5-136 and 162-163.   Naturally occurring TRAIL variants are also encompassed by the present invention. This Examples of such mutants include alternative mRNA splicing ligation. From the fruit (since TRAIL is encoded by a multi-exon gene) or TR A protein that results from proteolytic cleavage of the AIL protein, And the desired biological activity is retained. mRNA alternative splicing Can be partially truncated, such as naturally occurring soluble proteins. A biologically active TRAIL protein can be produced. Proteolysis Mutations include, for example, one or more ends from the TRAIL protein. Expression in different host cell types by proteolytic removal of terminal amino acids At the N- or C-terminus. In addition, proteolytic cleavage is Of TRAIL can be released from membrane-bound proteins. Allele Variants are also encompassed by the present invention.Oligomer   The invention relates to TRs in the form of oligomers, such as dimers, trimers or higher oligomers. AIL polypeptides. Oligomers include, for example, various TRAIL poly By disulfide bonds between cysteine residues on the peptide or TRAIL It can be formed by non-covalent interactions between polypeptide chains. In another embodiment Wherein the oligomer is a peptide residue fused to the TRAIL polypeptide 2-4 TRAIs bound by covalent or non-covalent interactions between L polypeptide. Such a peptide is a peptide linker (spacer ) Or a peptide having the property of promoting oligomerization. May be. Some polypeptides derived from leucine zippers and antibodies are listed below. As described in further detail, TRAIL polypeptides attached thereto Included in peptides that can promote the oligomerization of Preferably, TRAIL The polypeptide is soluble.   Fused to various parts of an antibody-derived polypeptide (including the Fc domain) The production of fusion proteins comprising a heterologous polypeptide is, for example, incorporated herein by reference. Ashkenazi et al. (PNAS USA 88: 10535,1991); Byrn et al. (Nature 344: 667,199). 0); and Hollenbaugh and Aruffo (“Construction of Immunoglobulin Fusion Proteins ", Current Protocols in Immunology, Appendix 4, 10.19.1-10 .19.11, 1992). In one embodiment of the present invention, TRAIL dimer is expressed by TRAIL against Fc partial polypeptide derived from antibody. Generated by fusing The term “Fc polypeptide” includes Natural and mutein forms, as well as hinge regions that promote dimerization Truncated Fc polypeptides containing Preferably, Fc poly Pe Peptides are fused to soluble TRAIL (eg, containing only the extracellular domain). Are combined.   Gene fusions encoding TRAIL / Fc fusion proteins can be expressed with appropriate expression Inserted into vector. The TRAIL / Fc fusion protein was very similar Allows the assembly of antibody molecules, resulting in interchain dislocation between the Fc polypeptides. A sulfide bond forms, resulting in bivalent TRAIL. In another embodiment, the TR AIL may be substituted for the variable region of an antibody heavy or light chain. The fusion protein As many as 4 extracellular TRAILs when produced using both the heavy and light chains of the antibody It is possible to form a TRAIL oligomer comprising a moiety.   One suitable Fc polypeptide is a native Fc partial polypeptide derived from human IgG1. A polypeptide, which is described in PCT Application No. WO 93/10151, which is incorporated herein by reference. Number. Another useful Fc polypeptide is described in US Pat. No. 5,457, No. 035, the Fc mutein. Of that mutein In the amino acid sequence, amino acid 19 is changed from Leu to Ala, and amino acid 20 is changed to Ala. Leu was changed to Glu, and amino acid 22 was changed from Gly to Ala. Except that it is identical to that of the native Fc sequence shown in WO 93/10151. This mutein Fc has reduced affinity for immunoglobulin receptors Is shown.   Alternatively, the oligomeric TRAIL may contain up to two TRAILs linked by a peptide linker. Or more soluble TRAIL polypeptides. Rice as an example Peptide linker described in National Patent No. 5,073,627, which is incorporated herein by reference. Is included. Multiple TRAIL polypeps separated by a peptide linker Tide-containing fusion proteins may be produced using conventional recombinant DNA techniques. Can be.   Another method for producing oligomeric TRAIL polypeptides is Leucindi. Requires the use of a pper. Leucine zipper domains are found in them Peptides that promote the oligomerization of proteins. Leucine Zipper is the first Have been identified in several DNA binding proteins (Landschulz et al., Science ce 240: 1759, 1988), which has since been found in a variety of different proteins . Some of the known leucine zippers are naturally occurring peptides and dimerize Or those derivatives that trimerize. Soluble oligomer TRAIL tamper Examples of leucine zipper domains suitable for producing proteins are incorporated herein by reference. PCT Application No. WO 94/10308. Dimerization in solution TRAIL polypeptide fused to a peptide to be trimmed or trimerized The recombinant fusion protein containing the peptide is expressed in a suitable host cell and The soluble oligomer TRAIL is recovered from the culture supernatant.   Some members of the TNF family of proteins are considered to exist in trimeric form. (Beutler and Huffel, Science 264: 667, 1994; Banner et al., Cell 7). 3: 431, 1993). Thus, trimeric TRAIL may benefit from enhanced biological activity. Can give points. Preferred leucine zipper moieties prefer trimers To form. One example is described in Hoppe et al. (FEBS Le tters 344: 191, 1994) and US patent application Ser. No. 08 / 446,922. Leucine zipper derived from the surfactant substance protein D (SPD). Naturally Other peptides derived from the present trimer protein produce trimeric TRAIL It can be used when doing.   Soluble FL expressed in CV-1 / EBNA cells as described in Example 7. The resulting oligomer formed spontaneously. This soluble FLA in the assay of Example 8 Increased, probably because the antibody crosslinked the TRAIL / receptor complex. This is because of the promotion. In one embodiment of the present invention, TRAIL biology Activity is achieved by using TRAIL with antibodies that can crosslink TRAIL. Increase. Cells to be killed include soluble TRAIL polypeptide and such Contact with both antibodies. An antibody fragment lacking the Fc portion is used. Bivalent forms of antibodies are separated by separate dimers It may be mixed or incubated with the AIL polypeptide.Expression system   The present invention relates to a recombinant expression vector for expressing TRAIL, and an expression vector for the same. A host cell transformed with the host cell. Use any suitable expression system Can be. Vectors can be derived from mammalian, microbial, viral or insect genes. Operably linked to appropriate transcriptional or translational regulatory nucleotide sequences, such as Encoding a TRAIL polypeptide, which is operably linked. Containing DNA. Examples of regulatory sequences include transcription promoters, operators Or enhancers, mRNA ribosome binding sites, and transcription and translation There are appropriate sequences to control the start and end. The nucleotide sequence is such that the regulatory sequence is T It is operably linked when it is operatively related to a RAIL DNA sequence. But Thus, the promoter nucleotide sequence is When controlling transcription of a TRAIL DNA sequence, Functionally coupled. An origin of replication that confers the ability to replicate in a desired host cell; And the selection genes that identify the transformed cells are generally included in the expression vector. It is.   In addition, sequences encoding the appropriate signal peptide may be included in the expression vector. May be included. The DNA sequence of the signal peptide (secretory leader) As TRAIL is first translated as a fusion protein containing the peptide, T It can be fused in frame to the RAIL sequence. A system that is functional in the intended host cell Gunal peptides promote extracellular secretion of TRAIL polypeptides. signal Peptides are cleaved from TRAIL polypeptide upon secretion of TRAIL from cells Is done.   Suitable host cells for expressing TRAIL polypeptide include prokaryotes, yeast and the like. Or higher eukaryotic cells. With bacterial, fungal, yeast and mammalian cell hosts Cloning and expression vectors suitable for use in are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York (1985) Are listed. The cell-free translation system is also a DNA construct disclosed herein. To Can be used to produce TRAIL polypeptides using derived RNA. Wear   Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or bacteria. Includes the genus Bacilli. Prokaryotic host cells suitable for transformation include, for example, For example, E. coli, Bacillus subtilis, Salmonella typhi phimurium), Pseudomonas, Streptomyces ( Contains various other species within the genus Streptomyces and Staphylococcus It is. In prokaryotic host cells such as E. coli, TRAIL polypeptides N-terminal methionine residue that promotes expression of a recombinant polypeptide in a prokaryotic host cell Groups can be included. N-terminal Met is expressed recombinant TRAIL polypeptide Can be cleaved from the tide.   Expression vectors for use in prokaryotic host cells are generally one or more. And more than one phenotype selectable marker gene. Phenotype selectable marker gene For example, proteins that confer antibiotic resistance or autotrophic requirements The encoding gene. Examples of expression vectors useful for prokaryotic host cells Commercially available such as cloning vector pBR322 (ATCC37017) Some are derived from functional plasmids. pBR322 comprises ampicillin and tet Has a gene for lacyclin resistance and thus identifies transformed cells Provide a simple means for Appropriate promoter and TRAIL DNA sequence The rows are inserted into the pBR322 vector. With other commercially available vectors For example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) And pGEM1 (Promega Biotec, Madison, WI, USA). .   Promoter sequences commonly used in recombinant prokaryotic host cell expression vectors Include β-lactamase (penicillinase), lactose promoter system ( Chang et al., Nature 275: 615,1978; and Goeddel et al., Nature 281: 544,1979). , Tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8: 4 And EP-A-36776) and the tac promoter (Maniatis, M. olecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p.412 , 1982). A particularly useful prokaryotic host cell expression system is the phage λP_Lpromotion Data -And cI857ts thermolabile repressor sequences are used. λP_Lpromoter Available from the American Type Culture Collection, which includes derivatives of Functional plasmid vectors include plasmid pHUB2 (E. coli strain JIM9, AT CC37092) and pPLc28 (E. coli RR1, ATCC53082) Is included).   TRAIL is alternatively present in yeast host cells, preferably in the genus Saccharomyces. (Saccharomyces) (eg, S. cerevisiae) be able to. Pichia or Kluyveromyces ) May be used. Yeast vectors are often 2μ yeast Origin of replication sequence from plasmid, self-replicating sequence (ARS), promoter region, Sequences for liadenylation, sequences for transcription termination and selectable marker inheritance Will have children. Suitable promoter sequences for yeast vectors include, in particular, meta- Rothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 2 55: 2073, 1980), or enolase, glyceraldehyde-3-phosphate dehydro Genase, hexokinase, pyruvate decarboxylase, Nase, glucose-6-phosphate isomerase, 3-phosphoglycerate , Pyruvate kinase, triosephosphate isomerase, Other glycolytic enzymes such as somerase and glucokinase (Hess et al., J. Adv. Enzyme Reg. 7: 149, 1968; and the promoter of Holland et al., Biochem. 17: 4900, 1978). Is included. Other suitable vectors and promoters for use in yeast expression are Hitzeman, EPA-73,657. Another option is Russ ell et al., J. Biol. Chem. 258: 2674, 1982) and Beier et al. (Nature 300: 724, 1982). ) Is the glucose repressible ADH2 promoter described by Yeast and Shuttle vectors that are replicable in both E. coli and E. coli DNA sequence from pBR322 (Amp^rGene and origin of replication) It can be constructed by insertion into a mother vector.   Yeast α-factor leader sequence regulates TRAIL polypeptide secretion Can be used. The α-factor leader sequence is often associated with a promoter sequence. Inserted between the structural gene sequence. For example, Kurjan et al., Cell 30: 933,1982. And Bitter et al., Proc. Natl. Acad. Sci. USA 81: 5330,1984. yeast Other leader sequences suitable for promoting secretion of the recombinant polypeptide from the host are , Known to those skilled in the art. The leader sequence has one or more restriction sites. May be modified near its 3 'end. This is the leader sequence It will promote fusion to structural genes.   Yeast transformation protocols are known to those skilled in the art. One such professional The protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75: 1929, 1978. Have been. The protocol of T. Hinnen et al.⁺About transformed cells Where the selection medium is 0.67% yeast nitrogen base, 0.5% Acid, 2% glucose, 10 μg / ml adenine and 20 μg / ml uracil Consists of   Yeast locus transformed by a vector containing the ADH2 promoter sequence Primary cells can be grown to induce expression in "rich" media. Examples of rich media include 80% 1% yeast extract, 2% peptone and 1% glucose. Consists of μg / ml adenine and 80 ug / ml uracil. A Derepression of the DH2 promoter occurs when glucose is depleted from the medium .   Mammalian or insect host cell culture systems also provide recombinant TRAIL polypeptides. Could be used for expression. Production of heterologous proteins in insect cells Baculovirus systems are described in Luckow and Summers, Bio / Technology 6:47 (198 Commented on in 8). Established cell lines of mammalian origin can also be used. Appropriate An example of a mammalian host cell line is the COS-7 line of monkey kidney cells (ATCC CR L1651) (Gluzman et al., Cell 23: 175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL163), Chinese hamster ovary (CHO) cells, Cells and the BHK (ATCC CRL10) cell line, and McMahan et al. (EMBO J. 10: 2821, 1991) derived from the African green monkey kidney cell line CVI. There is a CVI / EBNA cell line (ATCC CCL70).   The transcriptional and translational control sequences of the mammalian host cell expression vector contain the viral genome Could be cut out from Commonly used promoter sequences and And enhancer sequences include polyoma virus, adenovirus 2, Simian virus Derived from Lus 40 (SV40) and human cytomegalovirus. SV40 DNA sequences derived from the virus genome, for example, the early and late stages of SV40 origin. Motors, enhancers, splices and polyadenylation sites Used to provide other genetic elements for expression of a structural gene sequence in a host cell Can be The viral early and late promoters are both viral Easily from the viral genome as a fragment that can also have an origin of replication It is particularly useful because it can be obtained (Fiers et al., Nature 273: 113, 1978). Even smaller The larger or larger SV40 fragment also contains the SV40 viral origin of replication. About 250 bp extending from the HindIII site to the BglI site Can be used provided that the column is included.   Expression vectors for use in mammalian host cells are described, for example, in Okayama and Berg (Mol. Cell. Biol. 3: 280, 1983). Wear. C127 Stable high-level expression of mammalian cDNA in murine mammary epithelial cells A currently useful system is substantially as described by Cosman et al. (Mol. Immunol. 23: 935,1986). It can be constructed as described. By Cosman et al., Nature 312: 768,1984 The described high expression vector PMLSV N1 / N4 was designated as ATCC 39890. Has been deposited. Yet another mammalian expression vector is EP-A-0367566 and And WO 91/18982. Alternatively, the vector is retro It may be derived from a virus. Yet another suitable expression system is described in the Examples below. You.   One preferred expression system comprises Chinese hamster ovary (CHO) cells and An expression vector called PG5.7 is used. This expression vector is referred to herein. No. 08 / 586,509, filed Jan. 11, 1996, which is incorporated herein by reference. You. The PG5.7 component includes a fragment of CHO cell genomic DNA, followed by CM V-derived promoter followed by a sequence encoding the adenovirus tripartite leader. Sequence, followed by a sequence encoding dihydrofolate reductase (DHFR) Is included. These components are contained in the plasmid vector pGEM1 (Promega, Song, WI). TRAIL polypeptide (or containing TRAIL) DNA encoding the fusion protein) has a tripartite leader and DH It can be inserted between each sequence encoding FR. Approved in the field Me Add methotrexate to the media to increase expression levels as described be able to.   A fragment of CHO cell genomic DNA in vector PG5.7 Promotes L expression. Fragments of genomic DNA isolated from CHO cells The phage lysate contained was from American Type Culture on January 4, 1996. -Deposited with the Collection and given the deposit number ATCC97411. Be PG5.7 is a CHO genomic DNA insert in the strain deposit ATCC97411. G of nucleotides 8671 to 14507.   For expression of TRAIL, a native signal sequence, a heterologous signal sequence or Addition of a type II protein lacking a functional leader in mammalian host cells Wear. As an example, interleukin-7 (US Pat. No. 4,965,195) IL-7) signal sequence, described in Cosman et al., Nature 312: 768 (1984). Interleukin-2 receptor signal sequence; an interleukin-2 receptor described in EP367,566. -Leukin-4 receptor signal peptide; described in U.S. Patent No. 4,968,607 Type I interleukin-1 receptor signal peptide; and described in EP 460,846 There is a type II interleukin-1 receptor signal peptide listed.   A preferred expression system is a leader sequence derived from cytomegalovirus (CMV). Is used. Example 7 illustrates the use of one such reader. Example 7 No. 7, supra) and then the N-terminus of the soluble TRAIL polypeptide. Met Ala Arg Arg Leu Trp Ile Leu Ser Leu Leu Ala V al Thr Leu Thr Val Ala Leu Ala Ala Pro Ser Gln Lys Ser Lys Arg Arg Thr S er Ser (SEQ ID NO: 9). Array Residues 1-29 of No. 9 constitute the CMV-derived leader sequence, but residues 30-29 32 is an oligo used when constructing the expression vector described in Example 7. Encoded by nucleotides. In one embodiment, the poly His pep D encoding a tide (eg, a peptide containing six histidine residues) Located between the arrays.   An expression system using such a CMV-derived leader peptide is a system other than TRAIL. Useful for expressing proteins. Amino acids 1-29 of SEQ ID NO: 9 Provided herein is an expression vector comprising the DNA sequence to be loaded. Another fruit In an embodiment, the vector encodes amino acids 1-28 of SEQ ID NO: 9. Sequence. The DNA encoding the desired heterologous protein will serve as a leader It is located downstream of the encoding DNA and in the same reading frame. Yet another Of residues (eg, encoded by a linker or primer) As exemplified by the vector described in Example 7, the leader and the desired The DNA located between each sequence encoding the heterologous protein Can be loaded. As will be understood in the art, the expression vector comprises a leader and A promoter operably linked to the sequence encoding the heterologous protein; And any other desired regulatory sequences.   The leader peptide represented by SEQ ID NO: 9 is located after the arginine residue at position 29. It can be cleaved to yield the mature secreted form of the protein fused to it. Some Alternatively, or additionally, the cleavage is between amino acids 20 and 21 of SEQ ID NO: 9 or It can occur between amino acids 28 and 29.   One of skill in the art will recognize that one or more positions at which the signal peptide is cleaved can be used. Cell type, murine or human TRAIL to be expressed by the vector It will be appreciated that factors such as whether or not can vary. Compilation Analysis by a computer program showed that the major cleavage site was residue 20 of SEQ ID NO: 9. And 21 are possible. Between residues 22 and 23 and residues 27 and 2 It is anticipated that truncation between 8 and 8 is also possible. To illustrate, soluble murine TR Expression and secretion of AIL polypeptide is determined by CMV-derived signal pairs at a number of locations. Caused cleavage of the peptide. The three most prominent types of secreted proteins Is (in descending order) a truncation between amino acids 20 and 21 of SEQ ID NO: 9, amino acid 2 Cleavage between 2 and 23 and between amino acids 27 and 28 was caused.   A method for producing a heterologous recombinant protein includes transforming such an expression vector. A mammalian host cell under conditions that promote expression and secretion of the heterologous protein. And recovering the protein from the medium. CMV leader The expression systems used are, by way of example and not limitation, colony stimulating factors, Interferons, interleukins, other cytokines and cytokine receptors Can be used to produce any desired protein, including the body .Purified TRAIL protein   The invention can be produced by the above-described recombinant expression system or occurs naturally. Provide a purified TRAIL protein that can be purified from cells. The desired degree of purification is It may depend on the intended use of the protein. Relatively high purification, for example, This is desired when administering the protein in vivo. Advantageously, a TRAIL polypeptide Was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The protein band corresponding to the protein is purified so that it cannot be detected. Above As mentioned, due to differential glycosylation, variations in post-translational processing, etc. In addition, a number of bands corresponding to TRAIL protein were identified by SDS-PAGE. It will be understood by those skilled in the art that it can be detected. TRAIL protein In the sample, bands corresponding to different (non-TRAIL) proteins were not visible. Is considered to be purified to TRAIL is most preferably SDS-PA Purify to near homogeneity as indicated by a single protein band by GE analysis. Made. Protein bands were stained with silver, Coomassie blue, or (If the material is radioactively labeled) Can be.   One method of producing TRAIL protein encodes TRAIL TRAIL expresses host cells transformed with an expression vector containing a DNA sequence Culturing under the conditions as described. Next, culture TRAIL protein (From medium or cell extract). As those skilled in the art will appreciate, The procedure for purifying TRAIL depends on the type of host cell used and TRAIL. It may vary depending on factors such as whether secreted into the medium.   For example, when using an expression system that secretes a recombinant protein, Available protein concentration filters, such as Amicon or Millipore Pell icon The medium can be concentrated using an ultrafiltration device. After the concentration step, Can be applied to purification matrices such as gel filtration bases . Alternatively, an anion exchange resin such as diethylaminoethyl (DEAE) side group A matrix or support having the same can be used. The matrix is Lilamide, agarose, dextran, cellulose or protein purification And other types commonly used. Alternatively, using a cation exchange step Can be Suitable cation exchangers include sulfopropyl groups or carboxyl groups. There are various insoluble matrices containing boxymethyl groups. Sulfopropyl group is preferred Good. Finally, to further purify TRAIL, one or more reverse phases High Performance Liquid Chromatography (RP-HPLC) Bases (e.g., those having free methyl or other aliphatic side groups) Rica gel). Some or all of the above purification steps Can be used in various combinations to provide a purified TRAIL protein.   Recombinant protein produced in bacterial culture is first disrupted in host cells, centrifuged From the cell pellet for insoluble polypeptides or from soluble polypeptides. Extraction from the supernatant, followed by one or more concentration, salting out, ion exchange Isolated by affinity purification or size exclusion chromatography steps Can be Finally, RP-HPLC can be used as a final purification step. Wear. Microbial cells can be freeze-thaw cycles, sonication, mechanical disruption or cell lysis Destruction can be by any convenient method, including the use of sexual agents.   The transformed yeast host cells preferably have TRA as a secreted polypeptide. Used to express IL. This simplifies purification. Yeast host cell fermentation Secreted recombinant polypeptide from Urdal et al. (J. Chromatog. 296: 171, 1984). Can be purified by a method similar to that disclosed in US Pat. Urdal et al. Two sequential steps on a preparative HPLC column for purification of recombinant human IL-2 A reverse-phase HPLC step is described.   Alternatively, the TRAIL polypeptide is purified by immunoaffinity chromatography. Can be purified. Affiliates containing antibodies that bind TRAIL Column can be manufactured by conventional methods, and TRAIL is purified. It can be used when manufacturing. Example 4 was directed against TRAIL Methods for producing monoclonal antibodies are described.Properties and use of TRAIL   Programmed cell death (apoptosis) involves embryogenesis, metamorphosis, endocrine-dependent Occurs during woven atrophy, normal tissue turnover and death of immune thymocytes. Blog Regulation of rammed cell death is critical for the normal functioning of the immune system. An example As shown, T cells recognizing self-antigens were apoptotic during T cell maturation in the thymus. Other T cells are positively selected, although destroyed by the cis process. Some kind of self Some T cells that recognize the epitope (eg, disabled and given a given Self-protein determinants) escaped this elimination process and It has been suggested that it may play a role in autoimmune diseases (Gammon Et al., Immunology Today 12: 193, 1991).   Insufficient apoptosis was associated with certain symptoms, but high levels of apoptosis Cis cell death has been linked to other diseases. Apot for treating such diseases It is understood that it is desirable to determine and use agents that modulate lysis (Krom er, Advances in Immunology, 58: 211, 1995; Groux et al., J. Exp. Med. 175: 331, 1992. Sachs and Lotem, Blood 82:15, 1993).   The abnormal resistance of T cells to undergo apoptosis is associated with lymphocytosis, Lymphadenopathy, splenomegaly, accumulation of autoreactive T cells, development of autoimmune diseases, leukemia And the development of lymphomas (Kromer, supra; especially see pages 214-215) I want to.) Conversely, excessive T cell apoptosis leads to lymphopenia, systemic immunity. Has been suggested to play a role in epidemic deficiency and specific immunodeficiency But special cases related to infectious mononucleosis and cytomegalovirus infection Virus-induced immunodeficiency conditions, as well as tumor-mediated immunosuppression (Kromer, See above; especially page 214). CD4 in HIV infected individuals⁺T Cell loss is caused by inappropriate activation-induced cell death (AICD) due to apoptosis. (Groux et al., J. Exp. Med. 175: 331, 1992).   As demonstrated in Examples 5 and 8, TRAIL is a Jurkat clone Induces apoptosis in an acute T-cell leukemia cell line designated E6-1. But Thus, TRAIL may be used to study apoptosis, including the regulation of programmed cell death. It is a useful research reagent in research. Jurkat cells are leukemia derived from T cells Being a diseased cell line, the TRAIL of the present invention may TRAIL plays a role in apoptosis of other transformed T cells such as Used in studying the role of leap.   TRAIL binds Jurkat cells and further induces their apoptosis Lead. TRAIL can be obtained from freshly isolated murine thymocytes, or from healthy humans. Did not cause the death of freshly removed peripheral blood T cells (PBT) from the donor. Numerous applications arise from these properties of TRAIL.   The TRAIL polypeptide is a leukemia cell, or any TRAIL binding Can be used to purify other cell types. Leukemia cells, for example, Can be isolated from blood. In one embodiment, the cells are Affinity Chromatography binding TRAIL by tea chromatography -Purified using a matrix. For chromatography matrices Bound TRAIL is a full-length protein, TRAIL containing extracellular domain Fragment, TRAIL containing fusion protein or other described herein. A suitable TRAIL polypeptide. In one embodiment, the soluble The TRAIL / Fc fusion protein is loaded on a protein A or protein G column. On the other hand, binding by interaction between the Fc portion and protein A or protein G I do. Alternatively, TRAIL isolates leukemia cells by flow cytometry It can be used when doing.   The leukemia cells thus purified are thought to die after TRAIL binding. But the dead cells will still have cell surface antigens, and It can be used as an immunogen when eliciting blood antibody. Leukemia cells or The desired cell surface antigen isolated therefrom is further used in vaccine development. Can be   Because TRAIL binds and kills leukemia cells (Jurkat cell line) , TRAIL may also be useful in treating leukemia. The cure is white blood Contacting the diseased cells with an effective amount of TRAIL. In one embodiment The blood of a leukemia patient is contacted ex vivo with a TRAIL polypeptide. TR AIL may be immobilized on a suitable matrix. TRAIL is a leukemia cell Vesicle After binding, they are removed from the patient's blood and then the blood is returned to the patient.   Alternatively or additionally, the bone marrow isolated from the leukemia patient is replaced with leukemia cells in the bone marrow. Can be contacted with an effective amount of TRAIL to induce the death of TRAIL. Bone marrow For example, it is aspirated from the sternum or iliac crest and contacts leukemia cells with TRAIL. The vesicles can be removed. The bone marrow thus treated is returned to the patient.   TRAIL also binds to lymphoma and melanoma cells and Induce potosis (see Examples 5, 9 and 10). Therefore The use of TRAIL similar to that described above for leukemia cells, Applicable to tumor and melanoma cells. Why are TRAIL polypeptides restricted? Not used to treat cancer, including leukemia, lymphoma, and melanoma be able to. In one embodiment, the lymphoma is Burkitt's lymphoma . Table I in Example 9 shows that TRAIL was administered to several Burkitt lymphoma cell lines. To have a cytotoxic effect. Epstein-Barr virus It is the etiological agent of lymph node lymphoma.   TRAIL polypeptides are also used in treating viral infections. Contact with TRAIL causes death of cells infected with cytomegalovirus However, as described in Example 11, a similar cell type type when not infected was drawn. Did not wake up. TRAIL's ability to kill cells infected by other viruses Can be confirmed using the assay described in Example 11. Such a virus Is, but is not limited to, encephalomyocarditis virus, Newcastle disease virus Vesicular stomatitis virus, herpes simplex virus, adenovirus-2, bovine virus There are sexually transmitted diarrhea virus, HIV and Epstein-Barr virus.   An effective amount of TRAIL is administered to mammals suffering from viral infection, including humans Administer. In one embodiment, TRAIL is combined with interferon Used to treat viral infections. In the experiment described in Example 11, CMV Pretreatment of cells infected with γ-interferon is mediated by TRAIL Increased the lethality of infected cells. TRAIL is a cancer cell or viral It can be administered together with other drugs that show cytotoxic effects on the stained cells.   In another embodiment, the TRAIL is a cell preparation, tissue or transplant. Used to kill virus-infected cells in the affected organ. For example, bone Prior to transplanting the marrow into the recipient, the bone marrow is contacted with TRAIL and present therein. Virus-infected cells can be killed.   The TRAILs of the present invention are (directly) deficient or inadequate in quantity. Used to develop treatments for any disease mediated (directly or indirectly) be able to. A therapeutically effective amount of purified TRAIL protein can be used in such diseases Administer to patients suffering. Alternatively, the TRAIL DNA sequence may include such It can be used to develop gene therapy for the treatment of disease. Natural T The disclosure herein of the RAIL nucleotide sequence provides for the detection of a defective TRAIL gene. And its replacement with the normal TRAIL encoding gene. The missing gene Natural TRAIL disclosed in in vitro diagnostic assays, and herein Nucleotide sequence and TRA from patient suspected of being defective in this gene It can be detected by comparison with the sequence of the IL gene.   The present invention relates to purified TRAIL and a physiologically acceptable carrier, diluent or excipient. There is provided a pharmaceutical composition comprising the excipient. Suitable carriers, diluents and excipients are Is non-toxic at the dosages and concentrations used for Such compositions may be buffered Agents, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides Including proteins, amino acids, glucose, sucrose or dextrin Common in carbohydrates, chelating drugs such as EDTA, glutathione, and pharmaceutical compositions It may contain other commonly used stabilizers and excipients. Neutral buffered saline Or saline mixed with conspecific serum albumin, Included in a suitable diluent. The composition can be prepared with a suitable excipient solution (eg, sucrose). Can be formulated as a lyophilizate using diluent as a diluent.   For therapeutic use, the purified protein of the present invention can be administered to a patient, preferably a human, It is administered to treat the indicated condition in an appropriate manner. So, for example, drugs The pharmaceutical composition may be administered topically, by intravenous injection, continuous infusion, sustained release from an implant, or other suitable It can be administered by any technique. Appropriate dosages and frequency of administration are of course However, the nature and severity of the indicator condition being treated, the desired response, the condition of the patient, etc. Will depend on such factors.   The TRAIL protein used in the pharmaceutical composition is preferably TRAIL The protein is substantially free of other proteins of natural or endogenous origin; Desirably, it contains less than about 1% by weight of residual protein contaminants from the manufacturing process. It is purified so as not to be. Such compositions, however, may comprise stabilizers, carriers, It may contain other proteins added as excipients or co-therapeutics.   The TRAIL-encoding DNA disclosed herein, as reviewed above, Used in the manufacture of TRAIL polypeptide. TRAIL nucleotide sequence Row fragments are also useful. In one embodiment, such a flag is Fragments of the human or murine TRAIL DNA disclosed herein. At least about 17 contiguous nucleotides, more preferably at least 30 contiguous nucleotides Consecutive nucleotides. In the present specification, the DNA and the fragment And the RNA-complementary strand as a single strand of TRAIL DNA of SEQ ID NO: 1, 3 and 5. And provided in both double-stranded form.   Some uses of such TRAIL nucleic acid fragments include polymerase chain reaction. Use as a probe or primer in a reaction (PCR). One As an example, a probe corresponding to the extracellular domain of TRAIL can be used. You. Probes are suitable for in vitro assays and for Northern and Southern blots. It is used when detecting the presence of TRAIL nucleic acid by the method. Express TRAIL Cell type can also be identified. Such methods are well known and those skilled in the art A probe having an appropriate length can be selected according to a specific purpose application. P The CR contains 5 'and 3' promoters corresponding to the ends of the desired TRAIL DNA sequence. Using a primer, the sequence is isolated and amplified using conventional techniques.   Other useful fragments of TRAIL nucleic acids include target TRAIL mRNA (Sen). Or a single strand capable of binding to a TRAIL DNA (antisense) sequence Antisense or sense oligonucleotide containing nucleic acid sequence (RNA or DNA) Reotide. Such a fragment generally comprises at least about 14 nucleic acids. Nucleotides, preferably containing about 14 to about 30 nucleotides. Given Antisense or sense oligonucleotide based on the cDNA sequence of the The ability to produce reotide is described, for example, in Stein and Cohen, Cancer Res. 48: 2659. , 1988 and van der Krol et al., BioTechniques 6: 958,1988.   The binding of the antisense or sense oligonucleotide to the target nucleic acid sequence is Some, including accelerated degradation of the double helix, immature termination of transcription or translation Translation (RNA) or transcription (D Causes the formation of a double helix that blocks NA). Therefore, antisense Ligonucleotides can be used to block TRAIL protein expression it can.   The antisense or sense oligonucleotide may further comprise a modified sugar-phosphorus. Acid diester backbone (or other sugar linkages such as those described in WO 91/06629) Wherein the sugar linkage is endogenous. Resistant to sex nucleases. Such oligonucleotides containing resistant sugar linkages Otides are stable in vivo (ie, resistant to enzymatic degradation), but Sequence specificity so that it can bind to the target nucleotide sequence. Sen Other examples of antisense or antisense oligonucleotides are described in WO 90/10448. Organic residues such as those described, and target nucleic acid sequences such as poly (L-lysine) Covalent attachment to other residues that increase the affinity of the oligonucleotide for the sequence There are oligonucleotides that do. Still further, an intercalating agent such as ellipticin and Alkylating agents or metal complexes into sense or antisense oligonucleotides Binding to the antisense or sense oligonucleotide for the target nucleotide sequence. The binding specificity of the lignonucleotide can be altered.   Antisense or sense oligonucleotides include, for example, CaPO_FourIntermediary DNA transfection, electroporation, or Epsta Any gene transfer, including other gene transfer vectors such as invar virus Can be introduced into cells containing the target nucleic acid sequence. Preferred Alternatively, the antisense or sense oligonucleotide may be placed in a suitable retroviral vector. Insert a reotide and ligate the cell with a retrovirus containing the inserted sequence. Antibodies may be contacted in vivo or ex vivo with A sense or sense oligonucleotide is introduced into cells containing the target nucleic acid sequence. Suitable retroviral vectors include, but are not limited to, murine G Rovirus M-MuLV, N2 (retrovirus derived from M-MuLV) Or double-copy vectors designated DCT5A, DCT5B and DCT5C (See PCT application WO 90/13641). Or another promotion Oligonucleotides can be expressed using the promoter sequence.   Sense or antisense oligonucleotides are also described in WO 91/04753 As described above, the formation of a conjugate with a ligand binding molecule It can be introduced into cells. Suitable ligand binding molecules are limited Cell surface receptors, growth factors, other cytokines, or cells There are other ligands that bind to surface receptors. Preferably, the ligand binding moiety The binding of a ligand is a ligand binding component that binds to its corresponding molecule or receptor. Does not substantially interfere with the ability of the offspring or has sense or antisense oligonucleotides Inhibits little entry of leotide or its conjugates into cells.   Alternatively, sense or antisense oligonucleotides are described in WO 90/10448. As described, the formation of the oligonucleotide-lipid complex includes the target nucleic acid sequence. It can be introduced into cells. Sense or antisense oligonucleotide The do-lipid complex is preferably dissociated in the cell by endogenous lipase.Antibodies immunoreactive with TRAIL   The TRAIL proteins of the present invention or immunogenic fragments thereof may be antibodies Can be used. The present invention therefore provides TRAIL Provide an antibody that specifically binds, i.e., the antibody (confers non-specific binding). Binds to TRAIL via the antigen binding site of the antibody).   Polyclonal and monoclonal antibodies are produced by conventional techniques be able to. For example, Monoclonal Antibodies, Hybridomas: A New Dimension i n Biological Analyses, Kennet et al. (supervised), Plenum Press, New York (19 80); and Antibodies: A Laboratory Manual, Harlow and Land (supervised) , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, See NY, (1988). Monoclonal immunoreactive with TRAIL Antibodies are further illustrated in Example 4 below.   The antigen-binding function of such antibodies can be produced by conventional techniques. Fragments are also encompassed by the present invention. Example of such a fragment Include, but are not limited to, Fab, F (ab ') and F (ab' )_TwoThere are fragments. Antibody fragments produced by genetic engineering technology And derivatives are also provided.   The monoclonal antibodies of the present invention include chimeric antibodies, for example, murine monoclonals. Humanized forms of antibodies. Such humanized antibodies are produced by known techniques. And reduced immunogenicity when the antibody is administered to humans give. In one embodiment, the humanized monoclonal antibody is a murine individual Variable region (or just its antigen binding site) and constant region from human antibodies including. Alternatively, the humanized antibody fragment is an antigen of a murine monoclonal antibody Binding site and variable fragment from the human antibody (lacking the antigen binding site) May be included. Of chimeric and further engineered monoclonal antibodies Production methods include Riechmann et al. (Nature 332: 323, 1988) and Liu et al. (PNAS 84: 343 9,1987), Larrick et al. (Bio / Technology 7: 934,1989) and Winter and Ha rris (TIPS 14: 139, May 1993).   Some uses of antibodies include TRAIL polypeptides in vitro or in vivo. There are uses in assays to detect the presence of tide. Antibodies can also be used for affinity chromatography. Used when purifying TRAIL by chromatography.   Antibodies that can further block TRAIL binding to target cells are TR It can be used to inhibit the biological activity of AIL. Therapeutic methods are TR An effective amount of such an antibody in inhibiting AIL-mediated biological activity Administer n vivo. Therefore, directly or indirectly by TRAIL To treat diseases mediated by or worsened. Monoclonal antibodies are generally Preferred for use in such therapeutic methods.   Antibodies directed against TRAIL are useful for treating thrombotic microangiopathy It can be. One such disease is thrombotic thrombocytopenic purpura (TTP). (Kwaan, H.C., Semin. Hematol., 24:71, 1987; Thompson et al., Blood, 80: 1890, 1992). Increased mortality rates associated with TTP have been reported by the US Centers for Disease Control (the U.S.Cent ers for Disease Control) (Torok et al., Am. J. Hematol. 50:84, 1995). It has been tell.   Patients suffering from TTP (HIV⁺And HIV^-Plasma from patients (including patients) Induces apoptosis of human endothelial cells from microvessels, but not from large vessels No induction (Laurence et al., Blood, 87: 3245, April 15, 1996). Therefore, T The plasma of TP patients is one or more that directly or indirectly induces apoptosis. Is thought to have more factors. The assay described in Example 13 below Polyclonal antibody raised against TRAIL was Inhibited apoptosis induced by TTP plasma in vesicles. The data shown in Example 13 Data indicate that TRAIL is present in the serum of TTP patients and that microvascular endothelial cells Suggests that it may play a role in inducing apoptosis.   Another thrombotic microangiopathy is hemolytic uremic syndrome (HUS) (Moak e, J.L., Lancet, 343: 393, 1994; Melnyk et al., (Arch. Intern. Med., 155: 2077, 1995. Thompson et al., Supra). One embodiment of the present invention relates to Anti-TR to treat a condition often referred to as "adult HUS" It relates to the use of AIL antibodies. A disease known as childhood / diarrhea-related HUS The etiology is different from human HUS.   Other conditions characterized by small vessel thrombus formation are treated with anti-TRAIL antibodies Can be These symptoms include, but are not limited to: There is Cardiac problems seen in about 5-10% of pediatric AIDS patients are small vessel It is thought to be involved in thrombus formation. Failure of the microvasculature in the heart It has been reported in patients with keratosis. As another example, systemic lupus erythematosus (S LE) is contemplated.   In one embodiment, the patient's blood or plasma is treated with an anti-TRAIL antibody and ex Contact in vivo. Antibodies (preferably, monoclonal antibodies) are prepared by conventional methods. To a suitable chromatography matrix. The patient's blood Or plasma is chromatographic containing antibodies bound to a matrix After flowing through the column, it is returned to the patient. Immobilized antibody binds TRAIL As such, the TRAIL protein is removed from the patient's blood.   In another embodiment, the antibody is administered in vivo, where a blocking antibody is used. of Is desirable. Such antibodies inhibit TRAIL binding to target cells Identify using any suitable assay, such as by examining antibodies for performance can do. Alternatively, a blocking antibody may inhibit binding of TRAIL to target cells. It can be identified by assays for the ability to inhibit a biological effect. Example 12 Illustrates one suitable method of identifying a blocking antibody, wherein the antibody is Assayed for the ability of Jurkat cells to inhibit TRAIL-mediated lysis Is done.   The present invention therefore provides for the use of an effective amount of an antibody directed against TRAIL. Methods for treating thrombotic microangiopathy, including: The antibody of the present invention can be used in vivo Or used in ex vivo procedures to mediate TRAIL on microvascular endothelial cells. Mediated damage (eg, apoptosis) can be inhibited.   Anti-TRAIL antibodies may be used in conjunction with other drugs that are useful in treating certain diseases. Can be. Reported by Laurence et al. (Blood, 87: 3245, 1996) in  In vitro experiments, apoptosis mediated by TTP plasma of microvascular endothelial cells A slight reduction in serum was due to anti-Fas blocking antibody, aurin tricarboxylic acid, or cryoprecipitation. This was achieved by using depleted, depleted normal plasma.   Thus, patients undergo Fas-ligand-mediated apoptosis of endothelial cells. And TRAIL-mediated endothelial cell apoptosis It can be treated in combination with an agent. In one embodiment, the anti-TRA Both IL blocking antibodies and anti-FAS blocking antibodies are available from TTP or HUS It is administered to patients suffering from a disease characterized by thrombotic microangiopathy. Fas anti Examples of blocking monoclonal antibodies directed against protogen (CD95) are described herein. It is described in PCT Application Publication No. WO 95/10540, which is incorporated by reference.   Antibodies that are immunoreactive with TRAIL and a suitable diluent, excipient or carrier Provided herein are pharmaceutical compositions comprising the same. Suitable compositions of such compositions The components are as described above for the composition containing the TRAIL protein. You.   The following examples are given to illustrate specific embodiments of the present invention, and Should not be construed as limiting the scope of                   Example 1: Isolation of human TRAIL DNA   DNA encoding the human TRAIL protein of the present invention is obtained by the following procedure. Was isolated. The National Center for Biological  Information) (NCBI) TBLASTN search for dbEST data This was performed using the intervening sequence LVVXXXGLYYVYXQVXF (SEQ ID NO: 8). This array is Based on the most conserved part of the NF ligand series (Smith et al., Cell, 73: 1349,199 3). The expressed sequence tag (EST) file, GenBank accession number Z36726 , Using these search parameters. The GenBank file is T indicated that it was obtained from a human atrial cDNA library.   Two 30-b based sequences from the 3 'and 5' ends of this EST file p-oligonucleotide was synthesized. The oligonucleotide from the 3 'end has the sequence TGAAATCGAAAGTATGTTTGGGAATAGATG (nucleotides 636 to 665 of SEQ ID NO: 1) And the 5 'oligonucleotide comprises TGACGAAGAGAGTATGAACAGC CCCTGCTG (nucleotides 291 to 320 of SEQ ID NO: 1). Oligonuk Leotide is³²Labeling at the 5 'end with Pγ-ATP and polynucleotide kinase Was done. The two λgt10 cDNA libraries were labeled with these labeled oligonucleotides. Screening by conventional methods using an equimolar mixture of nucleotides as a probe. Was done. One library is a human heart 5 'extended cDNA library (St. ratagene Cloning Systems, La Jolla, CA). The other is as follows This was a manufactured peripheral blood lymphocyte (PBL) library. PBL is normal And 10 ng / ml of OKT3 (anti-CD3 antibody) and Treated with 10 ng / ml human IL-2 for 6 days. Wash the PBL cells and 500 ng / ml ionomycin (Calbiochem) and 10 ng / ml P Stimulated with MA for 4 hours. Messenger RNA was isolated from stimulated PBL cells. Released. The cDNA synthesized on the mRNA template was converted to a λgt10 phage vector.   The recombinant phage is plated on E. coli strain C600-HFL, and Screening was performed using standard plaque hybridization techniques. D The trocellulose filter is pulled from these plates in duplicate, and³² 60 mM Tris pH 8.0, 2 mM ED using P-labeled oligonucleotide TA, 5x Denhardt's solution, 6xSSC, 1mg / ml n-lauroyl sarco Syn, in a solution of 0.5% NP40 and 4 μg / ml SS salmon sperm DNA At 67 ° C. overnight. Next, filter 3xSSC And washed at 67 ° C. for 3 minutes.   Approximately 1 million positive plaques from heart 5 'elongated cDNA library Obtained from among plaques. This clone does not contain the 3 'end of the gene Was. Approximately 50 positive plaques were 500,000 using PBL Ripary Obtained from among plaques. Take 15 of these first round positive plaques And inserts from a rich pool to amplify phage inserts Amplification was performed using an oligonucleotide primer designed in the above. The resulting generation The products were resolved by 1.5% agarose gel electrophoresis and loaded on nitrocellulose. Blotted, and by standard Southern blot techniques,³²Labeled with P The obtained 30-mer oligonucleotide was used as a probe for analysis. Southern analysis Of the two plaques that produced the largest band in the second screening Thus, purified and isolated phage plaques were used as described above. Using the procedure described in   DNA from the isolated phage is produced by a plate lysis method, and The cDNA insert was excised with EcoRI and ligated in Tris borate EDTA buffer. (+) Ligation into plasmid. Next, these inserts are distributed by conventional methods. The sequences were sequenced and the resulting sequences were aligned.   The nucleotide sequence of human TRAIL DNA is shown in SEQ ID NO: 1, and The amino acid sequence encoded thereby is shown in SEQ ID NO: 2. This human TRAI The L protein has an N-terminal cytoplasmic domain (amino acids 1 to 18), Amino acids 19-38) and the extracellular domain (amino acids 39-281). This Has a calculated molecular weight of 32,508 daltons.   Escherichia coli strain transformed with the recombinant vector containing the TRAIL DNA DH10B cells were established on June 14, 1995 by American Type Culture. Deposited in the collection and given deposit number 69849. The deposit Made under the terms of the Dapest Treaty. The recombinant vector in the deposited strain is , Expression vector pDC409 (described in Example 5). The vector Complete code digested with SalI and NotI and shown in SEQ ID NO: 1 The human TRAIL DNA containing the region was ligated into the digested vector.      Example 2: Isolation of DNA encoding truncated TRAIL   DNA encoding the second human TRAIL protein was isolated as follows. . This truncated TRAIL induces apoptosis of Jurkat cells Does not show the ability to   PCR analysis was performed using the 30 mer described in Example 1 with the 5 'and 3' primers. 3 of the 14 first round plaque harvests in Example 1 It was shown to contain a short form of TRAIL DNA. From the short form of the gene oning Systems, La Jolla, CA) and sequenced.   The nucleotide sequence of this DNA is shown as SEQ ID NO: 3. Coded by it The amino acid sequence is shown in SEQ ID NO: 4. The encoded protein is N-terminal End cytoplasmic domain (amino acids 1-18), transmembrane portion (amino acids 19-38) and And the extracellular domain (amino acids 39-101).   The DNA of SEQ ID NO: 3 corresponds to nucleotides 359-50 of the DNA of SEQ ID NO: 1. 6 lacking the human TRAIL deletion mutant (huTRAILdv) clone Called The deletion causes a transposition in the reading frame, and as a result, An in-frame stop codon occurs after amino acid 101 of SEQ ID NO: 4. But Thus, the DNA of SEQ ID NO: 3 encodes a partially truncated protein. Amino acids 1-90 of SEQ ID NO: 2 are identical to amino acids 1-90 of SEQ ID NO: 4 is there. However, due to the deletion, the C-terminal of the huTRAILdv protein The terminal portions (amino acids 91-101 of SEQ ID NO: 4) correspond to the corresponding SEQ ID NO: 2 Different from the residue at the position.   huTRAILdv protein is a C-terminal member of the TNF family of proteins. It lacks the above conserved areas found at the edges. This huTRAILdv protein Quality cannot cause apoptotic death of Jurkat cells Further confirm the importance of these conserved regions with respect to biological activity.             Example 3: DNA encoding murine TRAIL   DNA encoding murine TRAIL was isolated by the following procedure. Baek CDNA line containing cDNA from mouse T cell line 7B9 in Bullies were produced as described by Mosley et al. (Cell 59: 335,1989). That la DNA from the library is filtered using conventional techniques on a nitrocellulose filter. Moved up.   Hybridization on the filter using a human TRAIL DNA probe Forming mouse cDNA was identified. Two separate probes are used for two rounds of scripting. Used in the training. Isolated and amplified using human TRAIL DNA as a template The broadened PCR reaction product, approximately 400 bp in length, is used for the first round of screening. Used as a probe in the probe. These PCR products were Region fragments. Used in second round screening The probe consisted of the complete coding region of human TRAIL DNA of SEQ ID NO: 1. Was. For use with random primed DNA labeling kit (Stratagene, La Jolla, CA) And the probe was radiolabeled.   After hybridization at 37 ° C. in 50% formamide, Washing was performed at 50 ° C. using 1 × SSC, 0.1% SDS. Both screenings The mouse cDNA that was positive in the round was isolated.   The nucleotide sequence of this DNA is shown in SEQ ID NO: 5, and thereby The encoded amino acid sequence is shown in SEQ ID NO: 6. The encoded protein is , N-terminal cytoplasmic domain (amino acids 1-17), transmembrane portion (amino acids 18-3) 8) and the extracellular domain (amino acids 39-291). This mouse TRA IL is 64% identical to human TRAIL of SEQ ID NO: 2 at the amino acid level is there. The coding region of the mouse TRAIL nucleotide sequence is the human 75% identical to the coding region of the nucleotide sequence                    Example 4: Antibodies that bind TRAIL   This example illustrates the production of a monoclonal antibody that specifically binds TRAIL. Testify. Suitable immunogens that can be used in raising such antibodies As such, but not limited to, purified TRAIL protein or its immunity Genomic fragments (eg, extracellular domains), containing TRAIL polypeptide Fusion proteins (eg, soluble TRAIL / Fc fusion proteins), and And cells expressing recombinant TRAIL on the cell surface.   Known techniques for producing monoclonal antibodies include U.S. Patent No. 4,411,993. There are those described in the issue. Simply put, complete Freund's adjuvant as immunogen Mice were immunized with TRAIL emulsified in bunt and 10-100 μl Inject subcutaneously or intraperitoneally in the amount of g. 10-12 days later, immunized subject The product is boosted with additional TRAIL milked in incomplete Freund's adjuvant. You. Thereafter, the mice are periodically immunized once or twice a week with an immunization schedule. Quarantine. Serum samples were subjected to a dot blot assay for TRAIL antibody or ELI. Retro-orbital bleeding or caudal tip for ZA (enzyme-linked immunosorbent assay) Collect periodically by end-to-end incision.   After detection of appropriate antibody titers, positive animals were given the last intravenous injection of TRAIL in saline. One injection is given. Three to four days later, the animals are sacrificed, spleen cells are harvested, and Spleen cells from NSI or, preferably, P3x63Ag8.653 (ATCC CRL158). Fused to a murine myeloma cell line such as 0). Fusion gives rise to hybridoma cells , In non-fused cells, myeloma hybrids and multi-stage microtiter plates. HAT (hypoxanthine, aminopte) inhibits the growth of Phosphorus and thymidine) in selective medium.   Hybridoma cells were subjected to ELISA for reactivity to purified TRAIL. In Engvall et al. (Immunochem. 8: 871,1971) and in US Pat. No. 4,703,004. Screen by adaptation of the disclosed technique. Positive hybridoma cells Intraperitoneal injection into syngeneic BALB / c mice to give high concentrations of anti-TRAIL Ascites containing null antibodies can be generated. Or hybridoma Cells are grown in vitro in flasks or roller bottles by various techniques. Can be made. Monoclonal antibodies raised in mouse ascites were treated with ammonium sulfate. Purification can be achieved by gel exclusion chromatography following the precipitation of chromium. Some Or affinity based on the binding of antibodies to protein A or protein G. Affinity chromatography based on binding to TRAIL It can be used in the same way as for chromatography.                  Example 5: DNA ladder apoptosis assay   About its ability to express human TRAIL and induce apoptosis Examined. The human TRAIL gene corresponds to the 3 ′ and 5 ′ ends of the coding region. The oligonucleotide is synthesized with SalI and N at the end of the oligonucleotide. An otI restriction site was included. The coding region of the human TRAIL gene was Amplified using standard PCR techniques with these oligonucleotides I let you. The PCR reaction products are digested with the restriction endonucleases SalI and NotI. After digestion, it was inserted into the vector pDC409 digested with SalI / NotI. pDC409 is an expression vector for use in mammalian cells, Can replicate in vesicles.   pDC409 is an expression vector designated pDC406 (MDC, incorporated herein by reference). cMahan et al., described in EMBO J. 10: 2821, 1991 and PCT application WO 91/18982. ). pDC406 contains SV40, Epstein-Barr virus and It has an origin of replication derived from pBR322 and is described by Dower et al., J. Immunol. 142: 4314 ( 1989) is a derivative of HAV-EO. pDC406 is HAV Deletion of an intron present in the adenovirus 23 tripartite leader sequence of EO It is different from HAV-EO. Multiple cloning sites (multiple restriction endonucleases) DNA (including thease cleavage site) into HIV and adenovirus It is transcribed and translated using the derived regulatory elements. The vector is also Has a gene that confers phosphorus resistance.   pDC409 had a deletion of the BglII site outside of the mecs, resulting in the deletion of the mecsBglII. It differs from pDC406 in that the sites are unique. Two Pme1 sites and One Srf1 site was added to mcs and three stop codons (TAG) Was placed downstream of the mcs to function in all three reading frames. DNA sequence A T7 primer / promoter was added to aid the decision step.   The monkey kidney cell line CV-1 / EBNA-1 (ATCC CRL10478) was obtained from McMahan Et al., Human CMV Intermediate-Early Enhancer / Promoter as described above. Epstein-Barr Virus Nuclear Antigen-1 (EBNA-1) CV-1 cell line using a gene encoding EBNA-1 that constitutively expresses (ATCC CRL70) induced by transfection. EBNA -1 gene is the episode of an expression vector such as pDC409 having an EBV origin of replication. Enable replication   CV1 / EBNA cells grown in Falcon T175 flasks were Transfection with "empty" pDC409 or pDC409 containing the human TRAIL coding region Was taken. The transformed cells are grown at 37 ° C. and 10% CO 2_TwoCulture for 3 days Nourished. Next, the cells are washed with PBS and incubated in 50 mM EDTA at 37 ° C. Incubate at 20 ° C for 20 minutes, scrape the flask with a cell scraper, and Washed once in PBS. Next, the cells were placed in 1% paraformaldehyde in PBS. At 4 ° C. for 10 minutes and washed three times in PBS.   Jurkat cells were used as target cells in this assay and TRAIL expression It was determined whether sex cells could induce their apoptosis. Jurkat fine Vesicular clone E6-1 was obtained from the American Type Culture Collection. A human acute T-cell leukemia cell line available under accession number ATCC TIB152 And described in Weiss et al. (J. Immunol. 133: 123-128, 1984). That jar Cut cells were treated with 10% fetal bovine serum and 10 μg / ml streptomycin. 200,000-5, cells in RPMI medium supplemented with and penicillin The cells were cultured to a density of 00000 / ml. 4 million cells / well Using 2.5 ml of medium in a 6-well plate, as indicated below Supernatant from fixed cells, cells transfected with Fas ligand And co-cultured with various combinations of various antibodies.   After 4 hours, cells are washed once in PBS and placed in a desktop centrifuge. At 1200 RPM for 5 minutes. Resuspend the pellet, and 10 mM Tris-HCl, 10 mM EDTA, pH 7.5 and 0.2% Incubation for 10 minutes at 4 ° C. in 500 μl of a buffer consisting of This lyses the cells but leaves the nuclei intact. Next, the lysate , Spin at 14,000 RPM for 10 minutes in a 4 ° C. microcentrifuge. Supernatant And remove 25: 24: 1 phenol-chloroform-isoamyla After extracting three times with 1 ml of alcohol, in the presence of 1 μg of glycogen carrier (Sigma) Was precipitated with NaOAC and ethanol.   The obtained pellet was mixed with 10 mM Tris-HCl, 10 mM EDTA, pH7. . 5 at 37 ° C. with 10 μg / ml RNase A. Incubated for minutes. Next, the DNA solution was added to a trisborate EDTA buffer solution. By electrophoresis on a 1.5% agarose gel and stained with ethidium bromide. , And photographed while transmitting ultraviolet light.   The results were as follows. Tracing with pDC409 or pDC409-TRAIL The transfected fixed CV1 / EBNA cells have a detectable DNA ladder. Did not occur. PDC409-TRA co-cultured with Jurkat cells IL-fixed cells developed a DNA ladder, but were co-cultured with Jurkat cells. No fixed pDC409 cells were generated.   The DNA ladder also contains a DNA encoding human Fas ligand in pDC409. Jars with concentrated supernatant from COS cells transfected with NA This was seen when cut cells were co-cultured. The supernatant is tamped from the cell surface. It is thought to contain soluble Fas ligand which is released by cytolysis. F The DNA ladder induced by the as ligand is a soluble shield directed against Fas. Can be blocked by adding 10 μg / ml of the monoclonal antibody. Came. This similar antibody was purified by Jurkat D by pDC409-TRAIL cells. The ladder of NA could not be inhibited because TRAIL was Cis is not induced.   In a similar assay, transfected with pDC409-TRAIL Fixed CV1 / EBNA cells resulted in a DNA ladder in U937 cells. U 937 (ATCC CRL1593) is a human histiocytic lymphoma cell line. Effek The target to target cell ratio was 1: 4 (assay for Jurkat target cells). As in the case of).   Fragmentation of cellular DNA into a pattern known as a DNA ladder Characteristic of apoptosis. In the above assay, TRAIL is a leukemia cell line. And induced apoptosis of lymphoma cell lines.                      Example 6: Northern blot analysis   TRAIL expression in a number of different tissue types was determined by conventional Northern blotting. Was analyzed. Poly A from various adult tissues⁺Northern blot containing RNA (Multiple tissue Northern blots I and II) were obtained from Clonetech (Palo Alto, CA). ). The other plots show that the RNA samples were 1.1% agarose-forma Resolve on Rudehid gel and apply H as directed by the manufacturer (Amersham Corporation). Blot on ybond-N and stain with methylene blue to determine RNA concentration. Created by monitoring. Blot shows the complete code for human TRAIL The region was probed with an antisense RNA riboprobe.   Human TRAIL mRNA is expressed in peripheral blood lymphocytes, colon, small intestine, ovary, prostate, breast It was detected in glands, spleen, placenta, lung, kidney, heart, pancreas and skeletal muscle. TRAI The L transcript is a large cell regression lymphoma cell line Karpas 299 (Fischer et al., Blood, 72: 234, 1988) and found to be abundant in tonsillar T cells. TRAIL message Di was slightly present in the Burkitt's lymphoma cell line called Raji.   TRAIL mRNA is detected in testis, brain or liver, as well as some T cell lines Was not done. TRAIL transcripts are not stimulated or have PMA and calcium Freshly isolated peripheral blood T cells (PBT) stimulated with umionophores for 20 hours Little or no detected in.               Example 7: Production of soluble TRAIL polypeptide   Soluble human TRAIL polypeptide comprising amino acids 95-281 of SEQ ID NO: 2 Was manufactured as follows. This polypeptide is a spacer moiety discussed above. Is a fragment of the extracellular domain lacking   An expression vector encoding soluble human TRAIL has the following amino acid sequence ( N-terminal to C-terminal), ie, human cytomegalovirus (C Fusion of in-frame DNA encoding amino acids 95-281 of TRAIL And Hopp et al. (Biotechnology 6: 1204-1210, 1988). To facilitate purification of the fused protein.   Isolating a TRAIL-encoding DNA fragment, and Encodes amino acids 95 to 281 of SEQ ID NO: 2 by ze-chain reaction (PCR) Oligonucleotide primer to determine the end of the DNA fragment And amplified. The 3 'primer has a NotI downstream of the TRAIL coding sequence. It was a 31-mer with additional sites. The 5 'primer has a TRAIL coding sequence This was performed by a conventional method using the above-mentioned human TRAIL cDNA as a template.   The reaction product is digested with SpeI and NotI and SalI and NotI Inserted into the expression vector pDC409 cut off with I (described in Example 5) . SalI-SpeI fragment encoding CMV open reading frame reader The annealed oligonucleotide that forms the DNA is also ligated into the vector. Was. The amino acid sequence of the CMV-derived leader is shown in SEQ ID NO: 9. SEQ ID NO: 9 Amino acids 1-29 are encoded by CMV DNA, ~ 32 correspond to the oligonucleotides used in constructing the vector. Loaded. E. coli cells are transfected with the ligation mixture and The desired recombinant expression vector was isolated therefrom.   CV1-EBNA cells (ATCC CRL10478; described in Example 5) Transfection with a recombinant vector called pDC409-Flag-shTRAIL ® down And cultured as described above. The culture supernatant is collected 3 days after transfection, I put it. Next, the column was washed with PBS. Monoclonal antibody M2 is available from Ho pp et al., supra, and Kodak Scientific Imaging Systems, New York. Available from Bun, Connecticut. Column using 50 mM citrate Eluted 800 μl fractions and immediately added 0.45 m 1M Tris (pH 8) Neutralized in 1 l. Adjust fractions to 10% glycerol and until needed Stored at -20 ° C. L was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Filtration analysis shows that the molecule is a multimer of approximately 80 kD in solution Suggest that. Without wishing to be bound by theory, the gel filtration analysis is acceptable. To indicate that the trimer was preferentially present. An expression vector called CD409-Flag-smTRAIL was constructed in a similar procedure. . DNA fragments encoding soluble murine TRAIL polypeptides It was isolated and amplified by PCR. Of the murine TRAIL sequence of SEQ ID NO: 6 Oligonucleotides for defining the ends of DNA encoding amino acids 99-291 Tides were used as 5 'and 3' primers in PCR.             Example 8: Lysis of leukemia cells by soluble TRAIL   In Example 5, the cells expressing human TRAIL were Jurkat cells (white Apoptosis of hematopoietic cell lines). In the next experiment, soluble human TRA IL polypeptide killed Jurkat cells.   Jurkat cells were transformed with 10% fetal bovine serum, 100 μg / ml streptomycin. Cultured in RPMI medium supplemented with syn and 100 μg / ml penicillin, The density was 200,000-500,000 cells / ml. The cells (96 50,000 cells / well in a volume of 100 μl in a well plate) Incubated with the reagents shown in FIG. 1 for 20 hours. "TRAIL supe. "Is transfected with pDC409-Flag-shTRAIL (see Example 7). Conditioned CV1 / EBNA cells from supernatant (10 μl / well) Means "Control supe." Indicates that the transfected C Supernatant from V1 / EBNA cells is meant. Where indicated, fixed anti After adding at a concentration and attaching overnight at 4 ° C or 2 hours at 37 ° C, the wells Was aspirated and washed twice with PBS to remove unbound antibody. Fas Riga Or M3 (Al), a blocking monoclonal antibody directed against Fas derson et al., J. Exp. Med. 181: 71, 1995; and PCT application WO 95/10540). Jurkat cells were included as indicated in the assay.   The metabolic activity of the cells treated in this way was determined by using alamar Blue The assay was performed by the following procedure by metabolic conversion of the dye. Alamar Blue Conversion Alama Leblue dye (Biosource International, Camarillo, CA) 10 μl / well And the optical density (OD at 550-600 nm) at the time the dye is added ) Was determined by subtracting from the OD 550-600 nm after 4 hours. No conversion of the dye is plotted as 0% viability and dye conversion in the absence of TRAIL The exchange concentration is plotted as 100% viability. % Viability is based on staining of experimental versus control cultures Calculated by multiplying the ratio by 100.   The results are shown in FIG. Error bars represent standard deviation of measurements from four separate wells. And their values are the average of these measurements.   TRAIL-containing supernatant causes significant decrease in viability of Jurkat cells did. A greater decrease in cell viability was due to TRAIL-containing supernatant and fixed Is to increase strength.   Fas ligand has shown the ability to kill Jurkat cells. Anti-Fas anti Body M3 inhibited Fas ligand activity but did not inhibit TRAIL activity Was.   Confirm that the change in dye conversion in the Alamar Blue assay was due to cell death To reduce the cell viability caused by TRAIL, trypanbub Confirmed by staining cells with roux.                 Example 9: Lysis of leukemia and lymphoma cells   In Examples 5 and 8, TRAIL was obtained from leukemia cell lines (Jurkat) and And induced apoptosis of a lymphoma cell line (U937). In the next experiment, 8 demonstrates the ability of TRAIL to kill leukemia and lymphoma cells.   The human cell line shown in Table I was prepared using 10% fetal calf serum, 100 μg / ml strain. Culture in RPMI medium supplemented with peptomycin and 100 μg / ml penicillin To a density of 200,000-500,000 cells / ml. The cell (50,000 cells / well in 100 μl volume in 96 well plate ) Was transformed with CV1 / transfected with pDC409-Flag-shTRAIL. Incubate for 20 hours with conditioned supernatant from EBNA cells (10 μl / well) I was vate.   Metabolic activity was determined by conversion of the alamar blue dye to the assay described in Example 8. The test was performed according to a standard procedure. The results are shown in Table I.   Confirm that the change in dye conversion in the Alamar Blue assay was due to cell death To reduce the cell viability caused by TRAIL, trypanbub Confirmed by staining cells with roux. Flick and Gifford (J. Immuno l. Methods 68: 167-175, 1984) Red staining also confirmed the results seen in the Alamar Blue assay. Cell death Potentiosis was determined by trivan blue staining and apoptotic fragmentation by microscopy. Confirmed by visualization of printouts.   As shown in Table I, a number of cancer cell lines respond to TRAIL-mediated killing. And was sensitive. Yet Another Specificity for TRAIL-Mediated Apoptosis Spore sensitivity can be confirmed using the assays described in this example. Wear.   TRAIL was raised against cell lines THP-1, K562, Karpas 299 and MP-1. Had little cytotoxic effect. K299 (also known as Karpas 299 DSM-ACC31) is a high-grade large cell regression lymphoma (Fischer et al., Blood, 72: 234). , 1988) was established from peripheral blood of males. MP-1 is spontaneously induced. EBV-transformed B cell line (Goodwin et al., Cell 73: 447, 1993). . Without wishing to be bound by theory, these four cell lines are Genes that may not express the receptor or inhibit apoptosis It may be characterized by up-regulation.    ^a Results are the mean ± SEM of 4 wells for each data point.                     Example 10: Cross-species activity of TRAIL   Interspecies cross-reactivity of human and murine TRAIL was examined as follows. Rat And human TRAIL with human melanoma cell line A375 (ATCC CRL1619). Incubated in the beginning. Because this is an adherent cell line, Rather, cell viability was determined using the crystal violet assay. A375 thin The vesicles were treated with 10% fetal calf serum, 100 μg / ml streptomycin and 100% The cells were cultured in DMEM supplemented with μg / ml penicillin. The cells (96 wells 10,000 cells / well in a volume of 100 μl in a plate) Incubated with soluble murine TRAIL as described in Example 7 for 72 hours . Crystal violet staining was performed by Flick and Gifford (J. Immunol. 68: 167-175, 1984). The result is a rat and Human and murine TRA in that human TRAIL killed A375 cells. Both ILs have been shown to be active on these human cells.   Immortalized murine lines to determine the ability of human TRAIL to act on murine cells The examination was performed using the fibroblast cell line L929. Human or murine TRAIL of L929 cells Incubation with causes a drop in crystal violet staining In human and murine TRAIL, murine cells (induced apoptosis) It was shown to be active. In addition to crystal violet, cell death Confirmed by Livan blue staining.                     Example 11: Lysis of CMV infected cells Has a cytotoxic effect on virus-infected cells.   Normal human gingival fibroblasts were plated on a 24-well plate with 10% CO2._Two And 10% fetal calf serum, 100 μg / ml streptomycin and 10% Grow to confluence in DMEM medium supplemented with 0 μg / ml penicillin. Fibroblast samples were processed as shown in FIG. Cytokine concentration , IL was 30 ng / ml. All samples given TRAIL also had TRAIL A double weight excess was given.   Pretreatment of cells with the indicated cytokines was for 20 hours. Cell site To infect megalovirus (CMV), the medium is aspirated and the cells are CMV was infected at an approximate MOI (multiple of infection) of 5 in DMEM. 2 o'clock After a while, replace the virus-containing medium with DMEM and add cytokines as indicated. I got it. After 24 hours, cells were stained with crystal violet dye as described ( Flick and Gifford, 1984, supra). Wash stained cells twice with water, 2% Rupture with 200 μl of sodium deoxycholate, dilute 5 times with water, and An OD at 570 nm was obtained. The maximum staining% is based on the sample that showed the strongest staining. Calculated by normalizing OD. Similar results, but some independent Obtained from experiments.   The results shown in FIG. 2 indicate that TRAIL specifically identified fibroblasts infected with CMV. To indicate that he was killed. This cell death is induced by pretreatment of cells with γ-interferon. Promoted by the law. Fibroblasts not infected with the virus almost die The absence was due to contact with TRAIL.                    Example 12: Assay to identify blocking antibodies   Blocking antibodies directed against TRAIL may be specific biological activities of TRAIL Can be identified by examining the antibody for its ability to inhibit. next In the assay, the monoclonal antibody was isolated from TRAIL-mediated Jurkat cells. The vesicles were examined for their ability to inhibit apoptosis. Jurkat cell line implemented This is described in Example 5. A hybridoma cell line producing the antibody was used in the assay. Hybridoma The supernatant from the culture was placed in RPMI complete medium in a 96-well microtiter plate. The RAIL fusion protein and the monoclonal antibody designated M2 were prepared in Example 7 It is described in.   Hybridoma supernatants were prepared at a 1:50 (v / v) dilution (starting concentration) and at Used in two-fold serial dilutions. 37 ° C, 10% CO_TwoIncubation for 30 minutes After incubation, add 50,000 Jurkat cells / well and incubate For 20 hours.   Next, cell viability was calculated by measuring the metabolic conversion of the alamar blue dye. The Alamar Blue Conversion Assay is described in Example 8. Monoclonal antibody It was found to inhibit tosis.                      Example 13: TRAIL blocking experiment   Skin-derived human microvascular endothelial cells are converted to thrombotic thrombocytopenic purpura (TTP) Plasma from patients or control plasma alone or with anti-TRAIL polyclonal Treated for 16-18 hours with serum in the presence. Use antiserum diluted 1: 2000 Was. Plasma was from two TTP patients denoted # 1 and # 2 below . The cells used in the assay were MVEC-1 (HMVEC 2753, Clonetics, San Francisco). Diego, CA) and MVEC-2 (DHMVEC 30282, Cell Systems, Kirkland, WA). Culture of these cells Nutrition can be maintained as described by Laurence et al. (Blood, 87: 3245, 1996). Wear.   The results were as follows. Data shown were stained with Providium iodide DNA histograms of the cells₀The peak is the apoptotic peak. (Oyaizu et al., Blood, 82: 3392, 1993; Nicoletti et al., J. Immunol. Metho ds, 139: 271, 1991; and Laurence et al., Blood, 75: 696, 1990).   The data show that plasma from TTP patients is apopotential for human microvascular endothelial cells. Induces tosis. This apoptosis is directed against TRAIL Inhibited by the polyclonal antibody.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), AU, CA, IL, J P, KR, MX, NO, NZ

Claims (1)

[Claims]   1. An isolated DNA encoding a TRAIL polypeptide, wherein said TR The AIL polypeptide comprises amino acids 1-281 of SEQ ID NO: 2 and SEQ ID NO: 6 An amino acid sequence selected from the group consisting of amino acids 1-291 of at least 80 % TRAIL polypeptide comprising an amino acid sequence that is The above isolated DNA capable of inducing apoptosis of vesicles.   2. The TRAIL polypeptide comprises amino acids 1-281 of SEQ ID NO: 2. And an amino acid sequence selected from the group consisting of amino acids 1-291 of SEQ ID NO: 6 The isolated DNA according to claim 1, comprising:   3. An isolated DNA encoding a soluble TRAIL polypeptide, comprising: The soluble TRAIL polypeptide is   (A) Extracellular domain of human TRAIL (amino acids 39-28 of SEQ ID NO: 2) 1); and   (B) a fragment of the extracellular domain An amino acid sequence that is at least 80% identical to a sequence selected from the group consisting of See; The soluble TRAIL polypeptide can induce apoptosis of Jurkat cells The above isolated DNA.   4. The soluble TRAIL polypeptide comprises:   (A) Extracellular domain of human TRAIL (amino acids 39-28 of SEQ ID NO: 2) 1); and   (B) a fragment of the extracellular domain, which is an apotato of Jurkat cell; The above fragment that can induce lysis The DNA according to claim 3, comprising an amino acid sequence selected from the group consisting of:   5. The soluble TRAIL polypeptide comprises amino acids x to 2 of SEQ ID NO: 2. 5. The method of claim 4, comprising 81 sequences, wherein x is an integer from 39 to 95. DNA.   6. The soluble TRAIL polypeptide comprises amino acid 95 to SEQ ID NO: 2. The DNA according to claim 5, comprising the sequence of 281.   7. The soluble TRAIL polypeptide comprises:   (A) Extracellular domain of human TRAIL (amino acids 39-28 of SEQ ID NO: 2) 1); and   (B) a fragment of the extracellular domain Comprising one or more conservative substitutions in an amino acid sequence selected from the group consisting of: ;   The conservedly substituted TRAIL can induce Jurkat cell apoptosis The DNA according to claim 3, which can be used.   8. An expression vector comprising the DNA according to any one of claims 1 to 7.   9. A method for producing a TRAIL polypeptide, comprising the steps of: Cultivated under conditions that promote TRAIL expression And recovering the TRAIL polypeptide.   10. A purified TRAIL polypeptide, comprising amino acids 1-2 of SEQ ID NO: 2. 81 and an amino acid selected from the group consisting of amino acids 1-291 of SEQ ID NO: 6 An amino acid sequence that is at least 80% identical to an acid sequence; The purified TRAIL polysaccharide wherein the peptide can induce apoptosis of Jurkat cells peptide.   11. Amino acids 1-281 of SEQ ID NO: 2 and amino acids 1-281 of SEQ ID NO: 6 The purified T of claim 10, comprising an amino acid sequence selected from the group consisting of: RAIL polypeptide.   12. CDNA insert of the recombinant vector deposited in strain ATCC 69849 A human TRAIL polypeptide encoded by the gene.   13. A purified soluble TRAIL polypeptide, comprising:   (A) Extracellular domain of human TRAIL (amino acids 39-28 of SEQ ID NO: 2) 1); and   (B) a fragment of the extracellular domain An amino acid sequence that is at least 80% identical to a sequence selected from the group consisting of See; The soluble human TRAIL polypeptide induces apoptosis of Jurkat cells A purified soluble TRAIL polypeptide as described above.   14． (A) Extracellular domain of human TRAIL (amino acid 39 of SEQ ID NO: 2) 281); and   (B) a fragment of the extracellular domain, which is an apotato of Jurkat cell; The above fragment that can induce lysis 14. The TRAIL polypeptide of claim 13, comprising an amino acid sequence selected from the group consisting of: Ripeptide.   15. Comprises the sequence of amino acids x to 281 of SEQ ID NO: 2, wherein x is 15. The TRAIL polypeptide of claim 14, which is an integer from 39 to 95.   16. 16. The TRA of claim 15, comprising amino acids 95-281 of SEQ ID NO: 2. IL polypeptide.   17． The soluble TRAIL polypeptide comprises:   (A) Extracellular domain of human TRAIL (amino acids 39-28 of SEQ ID NO: 2) 1); and   (B) a fragment of the extracellular domain Comprising one or more conservative substitutions in an amino acid sequence selected from the group consisting of: ;   The conservedly substituted TRAIL can induce Jurkat cell apoptosis 14. A TRAIL polypeptide according to claim 13,   18. An orifice comprising 2-3 soluble TRAIL polypeptides according to claim 13. Gommer.   19. A TRAI comprising three soluble TRAIL polypeptides of claim 16. L trimer.   20. Specifically binds the TRAIL protein according to claim 11 or 15 Antibodies.   21. 21. The antibody according to claim 20, wherein said antibody is a monoclonal antibody.